CN105164157A

CN105164157A - FC-receptor binding modified asymmetric antibodies and methods of use

Info

Publication number: CN105164157A
Application number: CN201480024044.6A
Authority: CN
Inventors: J·T·雷古拉; W·舍费尔; T·施洛特豪尔
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 2013-04-29
Filing date: 2014-04-25
Publication date: 2015-12-16
Also published as: PL2992010T3; RU2019108429A; US20160068613A1; JP6942162B2; JP2021138732A; US20200095310A1; RU2015145719A; JP2020015723A; US20200172607A1; JP7240443B2; EP3878866A1; ES2871383T3; MX2020003260A; US20220324955A1; US20160194389A1; RU2687043C2; EP2992010B1; CA2904805A1; WO2014177459A2; JP2016528166A

Abstract

Herein is reported an IgG class Fc-region comprising a first variant Fc-region polypeptide and a second variant Fc-region polypeptide, wherein the first variant Fc-region polypeptide is derived from a first parent IgG class Fc-region polypeptide and the second variant Fc-region polypeptide is derived from a second parent IgG class Fc-region polypeptide, whereby the first parent IgG class Fc-region polypeptide is identical to or different from the second parent IgG class Fc-region polypeptide, and the first variant Fc-region polypeptide differs from the second variant Fc-region polypeptide in one or more amino acid residues other than those amino acid residues in which the first parent IgG class Fc-region polypeptide differs from the second parent IgG class Fc-region polypeptide, and the IgG class Fc-region comprising the first variant Fc-region polypeptide and the second variant Fc-region polypeptide has an affinity to a human Fc-receptor that is different than that of an IgG class Fc-region comprising the first parent IgG class Fc-region polypeptide of a) and the second parent IgG class Fc-region polypeptide of a).

Description

The asymmetric antibody of the modification of FC-receptors bind and using method

Technical field

The present invention relates in its Fc-acceptor interaction, especially its FcRn interaction by the antibody of asymmetric modification and Fc district fusion polypeptide and using method thereof.

Background technology

Almost all Fc-acceptor all with the asymmetric combination in symmetrical Fc district of antibody.

Such as, the different aminoacids residue on people Fc γ-receptor II IA and Liang Tiao Fc district polypeptide chain interacts.Therefore, the sudden change (such as residue 233 to 238 place in lower hinge area) of asymmetric introducing needs the interaction that is used for increasing or reduce antibody and human Fc gamma receptor IIIA.

But, interaction between people's newborn Fc-acceptor FcRn is symmetrical: two FcRn molecules can with single IgG with 2:1 stoichiometry be combined (see, the people such as such as Huber, A.W., J.Mol.Biol.230 (1993) 1077-1083).Therefore, the sudden change of asymmetric introducing reduces and is bonded to the combination of a kind of FcRn/ by it, but does not reduce and be bonded to the combination of two kinds of FcRn/ by them.

The example of asymmetric IgG sample molecule include but not limited to by following technology or use following pattern to obtain those: the antibody of Triomab/Quadroma, Knobs-into-Holes, CrossMab, electrostatic coupling, LUZ-Y, chain exchange engineered constructs territory body, Biclonic and DuoBody.

In WO2012/125850, report the protein containing Fc, comprise asymmetric displacement in described protein Qi Fc district and there is increase with the keying action of people Fc γ-receptor II IA and the ADCC of enhancing active.

In WO2012/58768, report the separation heteromultimers comprising heterodimer Fc district, wherein heterodimer Fc district comprises variant CH3 structural domain, described variant CH3 structural domain is included in the amino acid mutation promoting heterodimer to be formed when stability increases, wherein heterodimer Fc district also comprises variant CH2 structural domain, and described variant CH2 structural domain comprises asymmetric amino acid modified to promote the selective binding effect of Fc γ acceptor.

In WO2011/131746, reporting by introducing asymmetric sudden change in the CH3 region of two monospecific initiation proteins, Fab-arm permutoid reaction can be forced to become directed and thus produce high stability heterodimeric protein.

The people such as Kim (Kim, H. people is waited, Invest.Ophthalmol.Vis.Sci.49 (2008) 2025-2029) report, except retinal pigment epithelium and tela chorioidea, ocular tissue, comprising ciliary body and iris, retina, conjunctiva, cornea, lens and optic tract, there is the FcRn transcript being in prediction size in display.Blood-ocular barrier display FcRn expression of receptor, shows that IgG may use this acceptor from ocular tissue to the transport of blood system.Due to interior ocular tissue as retina is isolated by blood-ocular barrier and blood system, by expectation, after intravitreal injection, only the short period of time cannot detect full length antibody in blood system.But the nearest pharmacokinetic data from monkey and people all shows Avastin in vitreum and occurs in blood in a few hours after intravitreal injection.Therefore, the FcRn function of receptors likely in conjunctiva lymphatic vessel will serve as outer row's acceptor effectively to eliminate Ag-Ab IgG mixture from conjunctiva lacuna.Although molecular weight is similar, in aqueous humor, detect IgG (150kDa), but IgA (160kDa) cannot be detected.Can by the existence to IgG selectively FcRn acceptor, explain that IgG and IgA is from the discordance between serum infiltrates to aqueous humor.

The people such as Kim report further people such as (, Mol.Vis.15 (2009) 2803-2812) Kim, H. directly intravitreal injection become the common approach to eye back segment delivery of therapeutic antibody portion for retinal disorder.The Avastin (lgG) of two kinds of intravitreal administrations and chicken IgY all cross internal limiting membrane barrier and diffuse into darker retinal structure.After diffusing through retina, Avastin strides across blood-retina barrier and is bled in systemic circulation.In retina, chicken IgY only locates along the side, nearly chamber of blood-retina barrier.In addition, choroidal artery exists for feminine gender to chicken IgY.After intravitreal administration, find that serum level that the physiology of Avastin is correlated with accounts at the most 30% of institute's injected dose.This prompting than previously recognize larger systemic side effects risk.Blood-ocular barrier represents and a kind ofly transports total length IgG and removed to the special mechanism in systemic circulation.This research of Kim confirms that this mechanism is the hypothesis of newborn Fc-acceptor.

In US2011/054151, report the composition and the method that are total to keying action for bivalent while antigen and unit price.

In US2011/236388, report dual specific bivalent anti-vegf/anti-ANG-2 antibody.

The antibody fusion protein that FcRn binding site is modified is reported in WO2010/121766.

The people such as Kim, J.K. report the location (Eur.J.Immunol.29 (1999) 2819-2825) in conjunction with the site of MHCI class associated receptor FcRn on human IgG.

The people such as Qiao, S.-W. report antibody-mediated antigen presentation and rely on FcRn (Proc.Natl.Acad.Sci.USA105 (2008) 9337-9342).

The people such as Kuo, T.T. report neonatal Fc receptor: from immunizing power to therapeutical agent (J.Clin.Immunol.30 (2010) 777-789).

The people such as Firan, M. report that MHCI class associated receptor FcRn plays in the maternal fetus gamma globulin transfer process of the mankind and must act on (Int.Immunol.13 (2001) 993-1002).

The people such as Vidarsson, G. report that FcRn is the IgG acceptor (Blood108 (2006) 3573-3579) phagocytic cell in phagocytosis with new role.

The people such as Gillies, S.D. report the validity (CancerRes.59 (1999) 2159-2166) by reducing the antibody-interleukin-22 fusion rotein caused by antibody-interleukin-22 fusion rotein and Fc-acceptor interaction.

Generation heterodimeric protein is reported in WO2013/060867.

General introduction

Have been found that the FcRn keying action of antibody or Fc district fusion polypeptide can be modified by the amino-acid residue changed in the non-corresponding position of Ge Tiao Fc district polypeptide, reason is that these changes jointly play a role in modification FcRn keying action.If antibody reported here and Fc district fusion polypeptide are useful, such as, be used for the treatment of the disease wherein requiring customization general retention time.

If reported here aspect is a kind of variant (people) IgG class Fc district comprising a Fc district polypeptide and the 2nd Fc district polypeptide,

Wherein

A) a Fc district polypeptide and the 2nd Fc district polypeptide are derived from identical parent (people) IgG class Fc district polypeptide, and

B) a Fc district polypeptide has at least different from the 2nd Fc district polypeptid acid sequence in a corresponding position according to KabatEUindex number system aminoacid sequences,

Thus with at a Fc district polypeptide compared with there is according to the corresponding position of KabatEUindex number system in the 2nd Fc district polypeptide (people) IgG class Fc district of (with (parent) people Fc district polypeptide a)) identical amino-acid residue, variant (people) IgG class Fc district has the different avidity to people Fc-acceptor.

Wherein

A) a Fc district polypeptide has at least different from the 2nd Fc district polypeptid acid sequence in a corresponding position according to KabatEUindex number system aminoacid sequences,

Thus, compared with having the IgG class Fc district of (with corresponding human Fc district) same amino acid residue with corresponding position in the first and second Fc district polypeptide, variant (people) IgG class Fc district has different from people Fc-receptor affinity.

Wherein

A) aminoacid sequence of a Fc district polypeptide is different in one or more amino-acid residue from the aminoacid sequence of the first parent IgG class Fc district polypeptide,

And

The aminoacid sequence of the 2nd Fc district polypeptide is different in one or more amino-acid residue from the aminoacid sequence of the second parent IgG class Fc district polypeptide, and

Thus, compared with the parent IgG class Fc district of the first and second parent IgG class Fc district polypeptide comprised a), variant (people) IgG class Fc district has the different avidity to people Fc-acceptor.

Wherein

A) aminoacid sequence of a Fc district polypeptide derived from first parent IgG class Fc district's polypeptide and the aminoacid sequence of the 2nd Fc district polypeptide derived from the second parent IgG class Fc district polypeptide, and

B) in a Fc district polypeptide and/or in the 2nd Fc district polypeptide, one or more sudden change is introduced, thus a Fc district polypeptide has at least different from the 2nd Fc district polypeptid acid sequence in a corresponding position according to KabatEUindex number system aminoacid sequences

Thus, compared with the IgG class Fc district of the first and second parent IgG class Fc district polypeptide comprised a), variant (people) IgG class Fc district has the different avidity to people Fc-acceptor.

In an embodiment in whole, variant (people) IgG class Fc district is the different dimerization Fc district of variant (people) IgG class.

In an embodiment in whole, first parent IgG class Fc district's polypeptide and the second parent IgG class Fc district polypeptide are non-human IgG class Fc district polypeptide.

In an embodiment in whole, first parent IgG class Fc district's polypeptide and the second parent IgG class Fc district polypeptide are identical IgG class Fc district polypeptide.

In an embodiment in whole, a Fc district polypeptide and the polypeptide pairing of the 2nd Fc district are to form the formation that dimerization (having function) Fc district causes heterodimer.

In an embodiment in whole, the first and second Fc district polypeptide are different from each other independently at least one amino-acid residue from respective parent IgG class Fc district polypeptide.

In an embodiment in whole, IgG class is selected from subclass IgG1, IgG2, IgG3 and IgG4.

In an embodiment in whole, people Fc-acceptor is selected from the newborn Fc-acceptor of people and human Fc gamma receptor.

In an embodiment in whole, a Fc district polypeptide is different from the 2nd Fc district polypeptide in 1 of the corresponding position according to KabatEUindex number system or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 amino-acid residues.

In one embodiment, compared with corresponding parent's human IgG class Fc district, variant (people) IgG class Fc district has staphylococcus (Staphylococcus) the albumin A keying action weakened.

In one embodiment, variant (people) IgG class Fc district has the SP keying action identical with corresponding parent's human IgG class Fc district.

In an embodiment in whole, variant (people) IgG class Fc district comprises both first and second Fc district polypeptide of human IgG1 or human IgG 4 subclass (derived from human source), and described polypeptide comprises to be selected from a Fc district polypeptide i) organizes I253A, H310A and H435A, or ii) organize H310A, H433A and Y436A, or iii) organize L251D, L314D and L432D, or iv) organize L251S, L314S and L432S (according to KabatEUindex number system numbering) one or two sudden change and comprise in the 2nd Fc district polypeptide be selected from sudden change L251D, L251S, I253A, H310A, L314D, L314S, L432D, L432S, H433A, one or two sudden change of H435A and Y436A (according to KabatEUindex number system numbering), thus the i) I253A that all suddenlys change, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S is all contained in variant (people) IgG class Fc district.

In an embodiment in whole, variant (people) IgG class Fc district comprises the first and second Fc district polypeptide of human IgG1 or human IgG 4 subclass (derived from human source), described polypeptide comprises sudden change I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D or L251S/L314S/L432S or its combination (according to KabatEUindex number system numbering) in Fc district, thus i) all sudden change all first or the 2nd in Fc district polypeptide, or ii) one or two sudden change in a Fc district polypeptide and one or two sudden change in the 2nd Fc district polypeptide, thus the i) I253A that all suddenlys change, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S is all contained in this Fc district.

In an embodiment in whole, variant (people) IgG class Fc district comprises the first and second Fc district polypeptide of human IgG1's subclass, wherein

A) both the first and second Fc district polypeptide also comprise sudden change L234A and L235A (according to KabatEUindex number system numbering), or

B) both the first and second Fc district polypeptide also comprise sudden change P329G (according to KabatEUindex number system numbering), or

C) both the first and second Fc district polypeptide also comprise sudden change L234A and L235A and P329G (according to KabatEUindex number system numbering), or

D) both the first and second Fc district polypeptide also comprise sudden change L234A and L235A (according to KabatEUindex number system numbering), and a Fc district polypeptide also comprises sudden change Y349C or S354C and mutation T 366W, and the 2nd Fc district polypeptide also comprise sudden change Y349C or S354C and mutation T 366S, L368A and Y407V, or

E) both the first and second Fc district polypeptide also comprise sudden change L234A and L235A and P329G (according to KabatEUindex number system numbering), and a Fc district polypeptide also comprises sudden change Y349C or S354C and mutation T 366W, and the 2nd Fc district polypeptide also comprises sudden change Y349C or S354C and mutation T 366S, L368A and Y407V.

In one embodiment, variant (people) IgG class Fc district comprises the first and second Fc district polypeptide of human IgG 4 subclass, wherein

A) both the first and second Fc district polypeptide also comprise sudden change S228P and L235E (according to KabatEUindex number system numbering), or

C) both the first and second Fc district polypeptide also comprise sudden change S228P and L235E and P329G (according to KabatEUindex number system numbering), or

D) both the first and second Fc district polypeptide also comprise sudden change S228P and L235E ((according to KabatEUindex number system numbering), and a Fc district polypeptide also comprises sudden change Y349C or S354C and mutation T 366W, and the 2nd Fc district polypeptide also comprise sudden change Y349C or S354C and mutation T 366S, L368A and Y407V

E) both the first and second Fc district polypeptide also comprise sudden change S228P and L235E and P329G (according to KabatEUindex number system numbering), and a Fc district polypeptide also comprises sudden change Y349C or S354C and mutation T 366W, and the 2nd Fc district polypeptide also comprises sudden change Y349C or S354C and mutation T 366S, L368A and Y407V.

If reported here aspect comprises antibody as variant (people) IgG class Fc district reported here or Fc district fusion polypeptide.

In one embodiment, antibody is monoclonal antibody.

In one embodiment, antibody is people's antibody, humanized antibody or chimeric antibody.

If reported here aspect is the nucleic acid of coding as variant (people) IgG class Fc district reported here.

If reported here aspect is the nucleic acid of coding as antibody reported here.

If reported here aspect is the nucleic acid of coding as Fc district fusion polypeptide reported here.

If reported here aspect comprises the host cell as nucleic acid reported here.

If reported here aspect is a kind of method produced as variant (people) IgG class Fc district reported here, described method comprises the host cell cultivated as reported here, thus produces variant (people) IgG class Fc district.

If reported here aspect is a kind of method produced as antibody reported here, described method comprises the host cell cultivated as reported here, thus produces antibody.

If reported here aspect is a kind of method produced as Fc district fusion polypeptide reported here, described method comprises the host cell cultivated as reported here, thus produces Fc district fusion polypeptide.

If reported here aspect is a kind of pharmaceutical preparation, it comprises as variant (people) IgG class Fc district reported here or as antibody reported here or as Fc district fusion polypeptide reported here.

As reported here aspect be used as medicine as variant (people) IgG class Fc district reported here or as antibody reported here or as Fc district fusion polypeptide reported here.

If reported here aspect is as variant (people) IgG class Fc district reported here or as antibody reported here or as Fc district fusion polypeptide reported here purposes in manufacture medicine.

As antibody reported here can be used as such as T cell recruiter, high as biological activity (effect) and remove fast from blood circulation (serum) Fc γ receptors bind thing, as there is quick scavenging(action) to reduce the antibody-drug conjugates of systemic side effects, or as front targeting antibodies.

Accompanying drawing is sketched

Fig. 1 has conceptual schematic view and the advantage of <VEGF-ANG-2>IgG1 or the IgG4 antibody of IHH-AAA sudden change (combination (EUindex according to Kabat numbers) of=sudden change I253A, H310A and H435A).

Fig. 2 is based on the viscosity measurement of small-scale DLS: at 200mM arginine/succinate, in the extrapolated viscosity (<VEGF-ANG-2> antibody VEGFang2-0016 (having IHH-AAA sudden change) compares with reference antibody VEGFang2-0015 (not having this kind of IHH-AAA to suddenly change)) of 150mg/mL in pH5.5.

Fig. 3 is at 20mM histidine buffering liquid, depend in 140mMNaCl, pH6.0 that the DLS of temperature assembles (comprise DLS and assemble starting temperature) (comparing as <VEGF-ANG-2> antibody VEGFang2-0016 (having IHH-AAA sudden change) reported here and reference antibody VEGFang2-0015 (not having this kind of IHH-AAA to suddenly change)).

Fig. 4 stores (main peak decline and high molecular (HMW) increase) on the 7th (compare the less gathering of display as <VEGF-ANG-2> antibody VEGFang2-0016 (having IHH-AAA sudden change) reported here and reference antibody VEGFang2-0015 (not having this kind of IHH-AAA to suddenly change)) at 100mg/mL at 40 DEG C.

The FcRn stable state avidity of Fig. 5 A and BA:VEGFang2-0015 (not having IHH-AAA to suddenly change) and B:VEGFang2-0016 (there is IHH-AAA sudden change).

The VEGFang2-0015 that Fig. 6 does not have IHH-AAA to suddenly change and the Fc γ RIIIa of the VEGFang2-0016 with IHH-AAA sudden change interact, and (the two is all the IgG1 subclass with P329GLALA sudden change in measurement; In contrast, anti-digoxin antibody (anti-Dig) and the antibody based on IgG4 of IgG1 subclass is used).

Fig. 7 A is for measuring schematic pharmacokinetics (the PK)-ELISA measuring principle of <VEGF-ANG-2> bi-specific antibody concentration in serum and full eye lysate.

Serum-concentration after Fig. 7 B intravenously (i.v.) applies: the VEGFang2-0015 not having IHH-AAA to suddenly change and the comparison with the VEGFang2-0016 that IHH-AAA suddenlys change.

Serum-concentration after applying in Fig. 7 C vitreum: the VEGFang2-0015 not having IHH-AAA to suddenly change and the comparison with the VEGFang2-0016 that IHH-AAA suddenlys change.

Fig. 7 DVEGFang2-0016 (having IHH-AAA sudden change) is at right eye and the palpebral fissure solution substrate concentration (compared with applying with intravenously, after being only exerted into right eye in vitreum) in left eye: only obvious concentration can be detected in right eye after applying in vitreum; After intravenously applies can not in eye lysate concentrations, reason is the low serum halflife of VEGFang2-0016 (have IHH-AAA sudden change).

Fig. 7 E can detect at the palpebral fissure solution substrate concentration of right eye with (compared with applying with intravenously, after being only exerted into right eye in vitreum) VEGFang2-0015 (not having IHH-AAA to suddenly change) in left eye: after applying in vitreum, in right eye, (with to a certain degree in left eye) detects the concentration of VEGFang2-0015; This display to diffuse to serum and from diffusing to left eye here, this can by the long half life explanation of VEGFang2-0015 (not having IHH-AAA suddenly change) from right eye; Obvious concentration can be detected in eye lysate at eyes after intravenously applies, reason is that the VEGFang2-0015 (not having IHH-AAA to suddenly change) of serum stable diffuses in eye.

Fig. 8 with reference to compared with wild-type (wt) antibody, half life (IHH-AAA suddenlys change) in half life (YTE suddenlys change) or the body that shortens in the body that the antibody of through engineering approaches shows prolongation in SPR analyzes in the ability that it is combined with FcRn, strengthen (YTE suddenlys change) or the keying action (IHH-AAA suddenlys change) weakened and in FcRn column chromatography, show the retention time strengthening or reduce; A) in single dose intravenous, the PK data after in applying 10mg/kg to huFcRn Transgenic male C57BL/6J mouse +/-276 are injected: the AUC data of the IgG that wild-type IgG and YTE and IHH-AAAFc-modifies; B) BIAcore sensing figure; C) FcRn affinity column wash-out; Wild-type anti-IGF-1 R antibodies (reference), the YTE-mutant of anti-IGF-1 R antibodies, the IHH-AAA-mutant of anti-IGF-1 R antibodies.

Fig. 9 depends on the number of the sudden change introducing Fc district, the change of retention time in FcRn affinity chromatography.

Figure 10 depends on the asymmetric distribution of the sudden change introducing Fc district, the change that FcRn combines.

Figure 11 is from the elution chromatography figure all in two heavy chains with the dual specific <VEGF-ANG-2> antibody (VEGF/ANG2-0121) of sudden change H310A, H433A and Y436A of twice continuous protein A affinity chromatography post.

Figure 12 is from the elution chromatography figure all in two heavy chains with the anti-IGF-1 R antibodies (IGF-1R-0045) of sudden change H310A, H433A and Y436A of protein A affinity chromatography post.

Immobilized albumin A on the <VEGF-ANG-2> antibodies CM5 chip that Figure 13 IgGFc district is modified.

The elution chromatography figure of Different L EssT.LTssT.LTVEGF-ANG-2> antibody on Figure 14 FcRn affinity column.

The combination of the different fusion polypeptide of Figure 15 and SP (SPR).

The combination of Figure 16 Different L EssT.LTssT.LTVEGF-ANG-2> antibody and anti-IGF-1 R antibodies mutant and immobilization albumin A (SPR).

The detailed description of embodiment of the present invention

I. define

Term " about " refers to +/-20% scope of hereafter numerical value.In one embodiment, term " about " refers to +/-10% scope of hereafter numerical value.In one embodiment, term " about " refers to +/-5% scope of hereafter numerical value.

For this paper object, " acceptor human framework " is such framework, it comprises and is derived from light variable domains (VL) framework of human normal immunoglobulin framework or people's consensus sequence framework or the aminoacid sequence of heavy-chain variable domains (VH) framework, as hereafter defined." be derived from " the acceptor human framework that human normal immunoglobulin framework or people have a framework and can comprise its identical aminoacid sequence, or it can change containing aminoacid sequence.In some embodiments, the number of amino acid change is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less or 2 or less.In some embodiments, VL acceptor human framework in sequence, have frame sequence with VL human normal immunoglobulin frame sequence or the mankind identical.

" affinity maturation " antibody refers to the antibody that there is one or more change in one or more hypervariable region (HVR), and does not have compared with these parental antibodies changed, and this kind of change causes antibody to improve the avidity of antigen.

Term " change " refers to sudden change (displacement) in parental antibody or fusion polypeptide (such as at least comprising the fusion polypeptide of the FcRn bound fraction in Fc district), inserts (interpolation) or lacks one or more amino-acid residue to obtain the antibody or fusion polypeptide modified." sudden change " refers to that the radical amino acid replacement of indication is different amino-acid residue to term.The L234A that such as suddenlys change refers to that the amino acid residue lysine of the 234th position in antibody Fc district (polypeptide) is replaced into amino-acid residue L-Ala (replacing L-Ala with Methionin) (according to EUindex numbering).

Term " amino acid mutation " refers to that at least one existing radical amino acid replacement is another different amino-acid residue (=replaceability amino-acid residue).Replaceability amino-acid residue can be " naturally occurring amino-acid residue " and be selected from L-Ala (three-letter codes: L-Ala (three-letter codes: ala, using single letter code: A), arginine (arg, R), l-asparagine (asn, N), aspartic acid (asp, D-Cys (cys, C), glutamine (gln, Q), L-glutamic acid (glu, E), glycine (gly, G), Histidine (his, H), Isoleucine (ile, I), leucine (leu, L), Methionin (lys, K), methionine(Met) (met, M), phenylalanine (phe, F), proline(Pro) (pro, P), Serine (ser, S), Threonine (thr, T), tryptophane (trp, W), tyrosine (tyr, and α-amino-isovaleric acid (val Y), V).Replaceability amino-acid residue can be " amino-acid residue that non-natural exists ".See such as US6,586,207, WO98/48032, WO03/073238, US2004/0214988, WO2005/35727, WO2005/74524, the people such as Chin, J.W., J.Am.Chem.Soc.124 (2002) 9026-9027; Chin, J.W. and Schultz, P.G., ChemBioChem11 (2002) 1135-1137; The people such as Chin, J.W., PICASUnitedStatesofAmerica99 (2002) 11020-11024; And Wang, L. and Schultz, P.G., Chem. (2002) 1-10 (whole document is complete all to be by way of reference incorporated to herein).

Term " aminoacid insertion " refers to that preposition place (interpolation) is incorporated at least one amino-acid residue in aminoacid sequence.In one embodiment, inserting will be insert one or two amino-acid residue.The amino-acid residue inserted can be the amino-acid residue that any naturally occurring or non-natural exists.

Term " aminoacid deletion " refers to that the pre-position in aminoacid sequence removes at least one amino-acid residue.

As used herein, term " ANG-2 " refers to human angiopoietin-2 (ANG-2) (being alternatively abbreviated as: ANGPT2 or ANG2) (SEQIDNO:31), it is such as at Maisonpierre, P.C. people is waited, Science277 (1997) 55-60 and Cheung, A.H. wait people, describe in Genomics48 (1998) 389-91.Find that Ang-1 (SEQIDNO:32) and-2 is as part people such as (, Nature407 (2000) 242-248) Yancopoulos, G.D. of the family tyrosine kinase Tie of blood vessel endothelium inside selective expression.The angiopoietin families member that current existence 4 kinds is determined.Angiogenin-3 and-4 (ANG-3 and ANG-4) can represent counterpart (people such as Kim, I., FEBSLet, 443 (1999) 353-356 of the divergence of homologous genes seat in mouse and the mankind widely; The people such as Kim, I., J.Biol.Chem.274 (1999) 26523-26528).ANG-1 and ANG-2 is accredited as agonist and antagonist respectively at first (about ANG-1, see the people such as Davis, S., Cell87 (1996) 1161-1169 in tissue culture experiments; And about ANG-2, see: the people such as Maisonpierre, P.C., Science277 (1997) 55-60).Main and the Tie2 (SEQIDNO:33) of all known angiogenin combines, and ANG-1 and-2 is all combined (Maisonpierre with 3nM (Kd) avidity with Tie2, P.C. people is waited, Science277 (1997) 55-60).

Term " antibody " uses with the widest meaning in this article and contains Multiple Antibodies works, include but not limited to monoclonal antibody, polyclonal antibody, multi-specificity antibody (such as, bi-specific antibody, three-specific antibody) and antibody fragment, as long as they demonstrate required antigen-and/or albumin A and/or FcRn-binding activities.

" with the antibody of reference antibody in conjunction with identical epi-position " refers to block reference antibody in competition assay and is combined the antibody reaching 50% or more with its antigen, and conversely, reference antibody blocks this antibody and is combined with its antigen and reaches 50% or more in competition assay.Exemplary competition assay is provided herein.

Term " asymmetric Fc district " refers to have a pair Fc district polypeptide of different aminoacids residue according to the corresponding position of KabatEUindex number system.

Term " the asymmetric Fc district about FcRn combines " refers to the Fc district that two polypeptide chains having different aminoacids residue by corresponding position form, wherein position is determined according to KabatEUindex number system, and thus different positions affects the combination of Fc district and newborn Fc-acceptor (FcRn) of people.For this paper object, in the asymmetric Fc district combined about FcRn Fc district two polypeptide chains between difference do not comprise the difference having introduced to promote to form different dimerization Fc district (such as generation of bi-specific antibody).These differences also can be asymmetrical, and namely two chains have difference at the non-corresponding amino-acid residue place according to KabatEUindex number system.These difference promotes different dimerization and reduces Homodimeric.The example of this kind of difference is so-called " tying into button " displacement (such as, see, US7695936 and US2003/0078385).Having been found that following knot in the wall scroll polypeptide chain in the IgG antibody Fc district of IgG1 subclass and buckling to change increases heterodimer and is formed: the Y407T 1) in a chain and the T366Y in another chain; 2) Y407A in a chain and the T366W in another chain; 3) F405A in a chain and the T394W in another chain; 4) F405W in a chain and the T394S in another chain; 5) Y407T in a chain and the T366Y in another chain; 6) T366Y and F405A in a chain and T394W and Y407T in another chain; 7) T366W and F405W in a chain and T394S and Y407A in another chain; 8) F405W and Y407A in a chain and T366W and T394S in another chain; With in 9 one chains) T366S, L368A and Y407V in T366W and another chain, that wherein finally lists is suitable especially.In addition, the change promotion heterodimer producing new disulphide bridges between Liang Tiao Fc district polypeptide chain is formed (see, such as, US2003/0078385).Have been found that the following displacement forming new intrachain disulfide bond in the wall scroll polypeptide chain of the cysteine residues of generation appropriate intervals for the IgG antibody Fc district of IgG1 subclass is formed to increase heterodimer: the Y349C in a chain and the S354C in another chain; , a Y349C in chain and the E356C in another chain; , a Y349C in chain and the E357C in another chain; , a L351C in chain and the S354C in another chain; , a T394C in chain and the E397C in another chain; Or the D399C in a chain and the K392C in another chain.Promote that other examples of the amino acid of different dimerization change are so-called " electric charge to displacement " (such as, see, WO2009/089004).Have been found that the following electric charge in the wall scroll polypeptide chain in the IgG antibody Fc district of IgG1 subclass increases heterodimer formation to displacement: K409D or K409E 1) in a chain and D399K or D399R in another chain; 2) K392D or K392E in a chain and D399K or D399R in another chain; 3) K439D or K439E in a chain and E356K or E356R in another chain; 4) K370D or K370E in a chain and E357K or E357R in another chain; 5) K409D and K360D in a chain adds D399K and E356K in another chain; 6) K409D and K370D in a chain adds D399K and E357K in another chain; 7) K409D and K392D in a chain adds the E357K in D399K, E356K and another chain; 8) K409D and K392D in a chain and the D399K in another chain; 9) K409D and K392D in a chain and D399K and E356K in another chain; 10) K409D and K392D in a chain and D399K and D357K in another chain; 11) K409D and K370D in a chain and D399K and D357K in another chain; 12) D399K in a chain and K409D and K360D in another chain; With 13) K409D and K439D in a chain and D399K and E356K on another chain.

Term " (with antigen) combines " refers in assay method in vitro, in one embodiment wherein antibody and surface bonding and by surperficial plasmon resonate (SPR) measure the combination of antibody and its antigen in the binding assay of antigen and antibodies.In conjunction with meaning in conjunction with 10 ^-8m or less, in some embodiments 10 ^-13to 10 ^-8m, in some embodiments 10 ^-13to 10 ^-9avidity (the K of M _d).

BIAcore assay method (GEHealthcareBiosensorAB, Uppsala, Sweden) research can be passed through combine.In conjunction with avidity by term k _a(association rate constants from the antibody of antibody/antigen mixture), k _d(dissociation constant) and K _d(k _d/ k _a) definition.

Term " is fitted together to " antibody and refers to a kind of antibody, and wherein a part for heavy chain and/or light chain is derived from particular source or species, and the remainder of heavy chain and/or light chain is derived from different sources or species.

Term " CH2 structural domain " refers to the part (the EU number system according to Kabat) extending to EU position 340 from EU position 231 of antibody heavy chain polypeptide.In one embodiment, CH2 structural domain has the aminoacid sequence of SEQIDNO:09: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQESTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK.

Term " CH3 structural domain " refers to the part extending to EU position 446 from EU position 341 of antibody heavy chain polypeptide.In one embodiment, CH3 structural domain has the aminoacid sequence of SEQIDNO:10: GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

" class " of antibody refers to the type of constant domain or the constant region had by its heavy chain.There is the antibody of five large classes: IgA, IgD, IgE, IgG and IgM, and several in these classifications can become subclass (isotype) by Further Division, such as, IgG ₁, IgG ₂, IgG ₃, IgG ₄, IgA ₁and IgA ₂.Heavy-chain constant domains corresponding to different classes of immunoglobulin (Ig) is called α, δ, ε, γ and μ.

Term " comparable length " refer to two peptide species comprise similar number amino-acid residue or can because of one or more and until maximum 10 bases acid residue and different in length.In one embodiment, Fc district polypeptide comprise similar number amino-acid residue or can be different because of 1 to 10 amino-acid residue.In one embodiment, Fc district polypeptide comprise similar number amino-acid residue or can be different because of 1 to 5 amino-acid residue.In one embodiment, Fc district polypeptide comprise similar number amino-acid residue or can be different because of 1 to 3 amino-acid residue.

Term " is derived from " and refers to that aminoacid sequence passes through in the introducing change of at least one position derived from parent amino acid sequence.Therefore, derivative aminoacid sequence is different from corresponding parent amino acid sequence at least one corresponding position (according to KabatEUindex number system antagonist Fc district numbering) place.In one embodiment, the aminoacid sequence being derived from parent amino acid sequence is different 1 to 15 amino-acid residue in corresponding position.In one embodiment, the aminoacid sequence being derived from parent amino acid sequence is different 1 to 10 amino-acid residue in corresponding position.In one embodiment, the aminoacid sequence being derived from parent amino acid sequence is different 1 to 6 amino-acid residue in corresponding position.Similarly, derivative aminoacid sequence and its parent amino acid sequence have homoamino acid sequence iden.In one embodiment, the aminoacid sequence being derived from parent amino acid sequence has the amino acid sequence identity of 80% or larger.In one embodiment, the aminoacid sequence being derived from parent amino acid sequence has the amino acid sequence identity of 90% or larger.In one embodiment, the aminoacid sequence being derived from parent amino acid sequence has the amino acid sequence identity of 95% or larger.

" effector function " refers to those biologic activity owing to antibody Fc district with antibody isotype variation.The example of antibody mediated effect subfunction comprises: the cytotoxicity (CDC) of C1q combination and Complement Dependent; Fc receptors bind; The cell-mediated cytotoxicity (ADCC) of antibody-dependant; Engulf; Lower cell surface receptor (such as B-cell receptor); Activate with B cell.

" significant quantity " of promoting agent (such as, pharmaceutical preparation) refers under the dosage and time period of necessity, effectively realizes the amount of required therapeutic or preventative result.

Term " Fc-fusion polypeptide " finger binding territory (such as antigen-binding domains is as single-chain antibody, or polypeptide is as the part of acceptor) is combined with the required target of display and/or albumin A combines and/or the fusions in the antibody Fc district of FcRn-binding activities.

Term " Ren Yuan Fc district " refers to the C end regions of people source heavy chain immunoglobulin, and it is at least containing some hinge area, CH2 structural domain and CH3 structural domain.In one embodiment, human IgG heavy chain Fc district is from Cys226 or the carboxyl terminal extending to heavy chain from Pro230.In one embodiment, Fc district has the aminoacid sequence of SEQIDNO:60.But the C in Fc district holds Methionin (Lys447) can exist or can not exist.Unless illustrated in addition herein, otherwise the numbering of the amino-acid residue in Fc district or constant region is according to such as Kabat, E.A. people is waited, SequencesofProteinsofImmunologicalInteres, the 5th edition, PublicHealthService, NationalInstitutesofHealth, Bethesda, MD (1991), EU number system described in NIHPublication913242, also referred to as EUindex.Fc district is made up of two heavy chain Fc district polypeptide, and described heavy chain Fc district polypeptide can be covalently bound each other by the hinge cysteine residue forming disulfide linkage between polypeptide.

Term " FcRn " refers to the newborn Fc-acceptor of people.FcRn plays the effect from lysosomal degradation pathway rescue IgG, causes clearance rate to reduce and half life increase.The heterodimeric protein that FcRn is made up of two polypeptide 50kDa major histocompatibility complex samples protein I class (α-FcRn) and 15kDa B2M (β 2m).FcRn combines with the CH2-CH3 in the Fc district of high-affinity and IgG part.Interaction between IgG and FcRn strictly relies on pH and occurs with 1:2 stoichiometry, namely an IgG is combined (Huber by its two heavy chains with two FcRn molecules, A.H. people is waited, J.Mol.Biol.230 (1993) 1077-1083).FcRn keying action acid pH (pH<6.5) in endosome occurs and IgG discharges at neutrophil cell surface (pH about 7.4).This interactional pH sensitive natur promotes that the IgG that FcRn mediate protection pinocytosis enters cell degrades by exempting from born of the same parents in endosome sour environment inside and receptors bind.FcRn assists IgG be recycled to cell surface and be released into blood flow when FcRn-IgG mixture is exposed to outside neutral pH environment subsequently subsequently.

Term " the FcRn bound fraction in Fc district " refers to the part of antibody heavy chain polypeptide, described part extends to EU position 261 from about EU position 243, and extend to EU position 293 from about EU position 275, and extend to EU position 319 from about EU position 302, and extend to EU position 348 from about EU position 336, and extend to EU position 393 and EU position 408 from about EU position 367, and extend to EU position 440 from about EU position 424.In one embodiment, the following one or more amino-acid residue F243 according to KabatEU numbering are changed, P244, P245P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, I336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440 (EU numbering).

" framework " or " FR " refers to the variable domains residue except hypervariable region (HVR) residue.The FR of variable domains is made up of following 4 FR structural domains usually: FR1, FR2, FR3 and FR4.Therefore, HVR sequence and FR sequence appear in VH (or VL) usually in the following sequence: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

Term " full length antibody " refers to such antibody, and described antibody has substantially similar to native antibody structure structure or has the heavy chain containing, for example Fc district defined herein.Full length antibody can comprise other structural domains, as scFv or scFab such as puted together with one or more chain of full length antibody.These conjugates are also contained by term full length antibody.

Term " heterodimer " or " different dimerization " refer to the molecule (such as having comparable length) comprising two polypeptide chains, wherein these two polypeptide chains have such aminoacid sequence, it has at least one different amino-acid residue in corresponding position, wherein corresponding position is determined according to the EUindex of Kabat.

Term " homodimer " and " homodimeric " refer to comprise that have can the molecule of two polypeptide chains of comparison length, and wherein these two polypeptide chains have the aminoacid sequence identical in corresponding position, and wherein corresponding position is determined according to the EUindex of Kabat.

If antibody reported here or Fc district fusion polypeptide can Jiu Qi Fc district be homodimeric or different dimerization, this determines relative to the sudden change paid close attention to or characteristic.Such as, (namely paying close attention to characteristic) is combined relative to FcRn and/or albumin A, Fc district (antibody) just suddenlys change H310A, H433A and Y436A is homodimeric (namely two heavy chain Fc district polypeptide all comprise these sudden changes) (paying close attention to these sudden changes with regard to the FcRn of Fc district fusion polypeptide or antibody and/or albumin A binding characteristic), but just suddenly change Y349C respectively simultaneously, T366S, L368A and Y407V (does not pay close attention to these sudden changes, because these sudden changes relate to heavy chain different dimerization and do not relate to FcRn/ albumin A binding characteristic) and (first group is only contained in a Fc district polypeptide with regard to sudden change S354C and T366W, and second group is only contained in the 2nd Fc district polypeptide) be different dimerization.Further such as, if Fc district fusion polypeptide reported here or antibody can be different dimerization (namely these sudden changes all relate to FcRn and/or the albumin A binding characteristic of dimer polypeptide) with regard to suddenly change I253A, H310A, H433A, H435A and Y436A, namely a Fc district polypeptide comprises sudden change I253A, H310A and H435A, and another Fc district polypeptide comprises sudden change H310A, H433A and Y436A.

Term " host cell ", " host cell system " and " host cell cultures " exchange and use and refer to the cell introducing exogenous nucleic acid wherein, comprise the offspring of this kind of cell.Host cell comprises " transformant " and " cell of conversion ", it cell comprising primary transformant and the filial generation derived from it, and no matter passage number is how many.Filial generation can be not identical with parental cell in nucleic acid content content, on the contrary can containing sudden change.Comprise mutant filial generation herein, described mutant filial generation has in the cell with initial conversion institute's filial generation of screening or selecting identical function or a biological activity.

" people's antibody " is so a kind of antibody, its have the antibody produced corresponding to people or people's cell aminoacid sequence or from utilizing the inhuman source of the sequence of people's antibody library or other encoding human antibody to derive.The humanized antibody comprising inhuman antigen binding residues is got rid of in this definition of people's antibody especially.

" mankind have framework " is such framework, the amino-acid residue that its representative the most often occurs in the selection of human normal immunoglobulin VL or VH frame sequence.Usually, the selection of human normal immunoglobulin VL or VH frame sequence is from the subgroup of variable domain sequence.Usually, sequence subgroup is as people such as Kabat, E.A., SequencesofProteinsofImmunologicalInterest, the 5th edition, BethesdaMD (1991), the subgroup described in NIHPublication91-3242,1-3 volume.In one embodiment, for VL, this subgroup is the subgroup κ I as described in the people such as Kabat above.In one embodiment, for VH, this subgroup is the subgroup III as described in the people such as Kabat above.

Term " people Fc district polypeptide " refers to the aminoacid sequence identical with " natural " or " wild-type " people Fc district polypeptide.Term " variant (people) Fc district polypeptide " refers to the aminoacid sequence derived from " natural " or " wild-type " people Fc district polypeptide because of at least one " amino acid change "." people Fc district " is made up of Liang Tiaoren Fc district polypeptide." variant (people) Fc district " is made up of Liang Tiao Fc district polypeptide, and wherein the two can be all variant (people) Fc district's polypeptide or Ge Shiren Fc district's polypeptide and another is variant (people) Fc district polypeptide.

In one embodiment, people Fc district polypeptide has human IgG 3Fc district's polypeptid acid sequence of human IgG1 Fc district's polypeptid acid sequence of SEQIDNO:60 or human IgG2 Fc district's polypeptid acid sequence of SEQIDNO:61 or SEQIDNO:62 or the human IgG 4Fc district polypeptid acid sequence of SEQIDNO:63.In one embodiment, variant (people) Fc district polypeptide derived from SEQIDNO:60 or 61 or 62 or 63 Fc district polypeptide and compared with SEQIDNO:60 or 61 or 62 or 63 Ren Fc district polypeptide, there is at least one amino acid mutation.In one embodiment, variant (people) Fc district polypeptide comprises/has the amino acid mutation from about 1 to about 12, and has about 1 to about 8 amino acid mutation in one embodiment.In one embodiment, variant (people) Fc district's polypeptide and SEQIDNO:60 or 61 or 62 or 63 Ren Fc district polypeptide have at least about 80% homology.In one embodiment, variant (people) Fc district's polypeptide and SEQIDNO:60 or 61 or 62 or 63 Ren Fc district polypeptide have at least about 90% homology.In one embodiment, variant (people) Fc district's polypeptide and SEQIDNO:60 or 61 or 62 or 63 Ren Fc district polypeptide have at least about 95% homology.

Variant (people) the Fc district polypeptide derived from SEQIDNO:60 or 61 or 62 or 63 Ren Fc district polypeptide is limited by contained amino acid change.Therefore, such as, term P329G refers to have proline(Pro) to derivative variant (people) the Fc district polypeptide of glycine mutation Ren Fc district polypeptide from relative to SEQIDNO:60 or 61 or 62 or 63 Ren Fc district polypeptide at amino acid position 329.

As used herein, whole constant region of heavy chain and light chain and the amino acid position of structural domain are according to people such as Kabat, SequencesofProteinsofImmunologicalInterest, 5th edition .PublicHealthService, NationalInstitutesofHealth, Bethesda, MD (1991)) in describe Kabat numbering system numbering and be called in this article " according to (Kabat) numbering ".Specifically, the people such as Kabat, SequencesofProteinsofImmunologicalInterest, 5th edition .PublicHealthService, NationalInstitutesofHealth, the Kabat number system (see 647-660 page) of Bethesda, MD (1991) for the light chain constant domain CL of κ and λ isotype and KabatEUindex number system (see 661-723 page) for constant heavy structural domain (CH1, hinge, CH2 and CH3).

Human IgG1 Fc district polypeptide has following aminoacid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：60).

The Fc district polypeptide that the human IgG1 Fc district with sudden change L234A, L235A derives has following aminoacid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：64).

The Fc district polypeptide that the human IgG1 Fc district with Y349C, T366S, L368A and Y407V sudden change derives has following aminoacid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：65).

The Fc district polypeptide that the human IgG1 Fc district with S354C, T366W sudden change derives has following aminoacid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：66).

The Fc district polypeptide that the human IgG1 Fc district with L234A, L235A sudden change and Y349C, T366S, L368A, Y407V sudden change derives has following aminoacid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGF

YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：67).

There is L234A, L235A and S354C, Fc district polypeptide that the human IgG1 Fc district of T366W sudden change is derivative have following aminoacid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：68).

The Fc district polypeptide that the human IgG1 Fc district with P329G sudden change derives has following aminoacid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：69).

The Fc district polypeptide that the human IgG1 Fc district with L234A, L235A sudden change and P329G sudden change derives has following aminoacid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：70).

The Fc district polypeptide that the human IgG1 Fc district with P239G sudden change and Y349C, T366S, L368A, Y407V sudden change derives has following aminoacid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：71).

The Fc district polypeptide that the human IgG1 Fc district with P329G sudden change and S354C, T366W sudden change derives has following aminoacid sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：72).

There is L234A, L235A, P329G and Y349C, Fc district polypeptide that the human IgG1 Fc district of T366S, L368A, Y407V sudden change is derivative have following aminoacid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：73).

The Fc district polypeptide that the human IgG1 Fc district with L234A, L235A, P329G sudden change and S354C, T366W sudden change derives has following aminoacid sequence:

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

CKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN

VFSCSVMHEALHNHYTQKSLSLSPGK(SEQIDNO：74).

Human IgG 4Fc district polypeptide has following aminoacid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：63).

The Fc district polypeptide that the human IgG 4Fc district with S228P and L235E sudden change derives has following aminoacid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：75).

The Fc district polypeptide that the human IgG 4Fc district with S228P, L235E sudden change and P329G sudden change derives has following aminoacid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：76).

The Fc district polypeptide that the human IgG 4Fc district with S354C, T366W sudden change derives has following aminoacid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：77).

The Fc district polypeptide that the human IgG 4Fc district with Y349C, T366S, L368A, Y407V sudden change derives has following aminoacid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：78).

There is S228P, L235E and S354C, Fc district polypeptide that the human IgG 4Fc district of T366W sudden change is derivative have following aminoacid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：79).

There is S228P, L235E and Y349C, Fc district polypeptide that the human IgG 4Fc district of T366S, L368A, Y407V sudden change is derivative have following aminoacid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：80).

The Fc district polypeptide that the human IgG 4Fc district with P329G sudden change derives has following aminoacid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：81).

There is P239G and Y349C, Fc district polypeptide that the human IgG 4Fc district of T366S, L368A, Y407V sudden change is derivative have following aminoacid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：82).

The Fc district polypeptide that the human IgG 4Fc district with P329G and S354C, T366W sudden change derives has following aminoacid sequence:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：83).

There is S228P, L235E, P329G and Y349C, Fc district polypeptide that the human IgG 4Fc district of T366S, L368A, Y407V sudden change is derivative have following aminoacid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：84).

There is S228P, L235E, P329G and S354C, Fc district polypeptide that the human IgG 4Fc district of T366W sudden change is derivative have following aminoacid sequence:

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY

KCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVK

GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN

VFSCSVMHEALHNHYTQKSLSLSLGK(SEQIDNO：85).

Hereafter show the comparison result (EU numbering) in different people Fc district:

" humanization " antibody refers to the chimeric antibody comprised from the amino-acid residue of non-human HVR and the amino-acid residue from people FR.In certain embodiments, humanized antibody will comprise at least 1 and general 2 variable domains substantially whole, wherein all or substantially whole HVR (such as, CDR) corresponding with those HVR of non-human antibody, and all or substantially whole FR corresponding with those FR of people's antibody.Humanized antibody optionally can comprise the antibody constant region that derives from people's antibody at least partially." the humanization form " of antibody such as, non-human antibody, refers to the antibody experiencing remarkable source.

As used herein, term " hypervariable region " or " HVR " refer to following each region in antibody variable knot territory, described region is high in sequence to be become (" complementary determining region " or " CDR ") and forms the ring (" Gao Bianhuan ") that structure is determined, and/or containing antigen contact residue (" antigen contact ").Usually, antibody comprises six HVR; In VH in three (H1, H2, H3) and VL three (L1, L2, L3).HVR as mentioned herein comprises

A () appears at the Gao Bianhuan (Chothia at 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2) and 96-101 (H3) amino-acid residue place, and Lesk C., A.M., J.Mol.Biol.196 (1987) 901-917);

B () appears at the CDR (Kabat at 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) amino-acid residue place, E.A. people is waited, SequencesofProteinsofImmunologicalInterest, 5th edition, PublicHealthService, NationalInstitutesofHealth, Bethesda, MD (1991), NIHPublication91-3242);

C () appears at the antigen contact (people such as MacCallum, J.Mol.Biol.262:732-745 (1996)) at 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2) and 93-101 (H3) amino-acid residue place; With

D the combination of () (a), (b) and/or (c), comprises HVR amino-acid residue 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

In one embodiment, HVR residue comprises those of the qualification of elsewhere in this specification sheets.

Unless otherwise indicated, otherwise other residues (such as, FR residue) in HVR residue and variable domains are in this article according to KabatEUindex number system that (people such as Kabat, numbers above).

As used herein, term " IGF-1R " refers to any natural IGF-1R from any vertebrate origin, wherein unless otherwise indicated, otherwise described vertebrate origin comprise Mammals as primates (such as, the mankind) and rodents (such as, Mouse and rat).This term is contained " total length ", unprocessed IGF-1R and any type of IGF-1R that produces because of processing in cell.Naturally occurring IGF-1R variant also contained in this term, such as, and splice variant or allelic variant.The aminoacid sequence of people IGF-1R shows at SEQIDNO:11.

" individuality " or " object " is Mammals.Mammals includes but not limited to domesticate animals (such as, milk cow, sheep, cat, dog and horse), primates (such as, people and non-human primates are as monkey), rabbit and rodents (such as, Mouse and rat).In certain embodiments, individual or to as if people.

" separation " antibody be with a kind of antibody of the Component seperation of its physical environment.In some embodiments, by antibody purification to being greater than 95% or 99% purity, as by such as electrophoresis (such as, SDS-PAGE, isoelectrofocusing (IEF), capillary electrophoresis) or chromatogram (such as, size exclusion chromatogram or ion-exchange or reversed-phase HPLC) determined.About the summary of the method for assessment antibody purity, see, such as, the people such as Flatman, S., J.Chrom.B848 (2007) 79-87.

" separation " nucleic acid refer to the nucleic acid molecule of the Component seperation of its physical environment.The nucleic acid be separated comprises and is contained in intracellular nucleic acid molecule, and described cell is usually containing this nucleic acid molecule, but this nucleic acid molecule is present in outer or different from its native chromosomal sites chromosome position place of karyomit(e).

The isolating nucleic acid of anti-IGF-1 R antibodies " coding " refers to one or more nucleic acid molecule of encoding antibody heavy and light chain (or its fragment), is included in this kind of nucleic acid molecule in single carrier or independent carrier and one or more positions exist in host cell this kind of nucleic acid molecule.

" monoclonal antibody " refers to the antibody that basically homogeneous antibody population obtains as used herein, the term, namely, the single antibody forming this colony is identical and/or in conjunction with identical epi-position, except possible variant antibodies (such as, occur containing naturally occurring sudden change or during generation monoclonal antibody preparations), this kind of variant exists with small amount usually.Contrary from the polyclonal antibody preparations generally comprised for the different antibodies of different determinant (epi-position), often kind of monoclonal antibody of monoclonal antibody preparations is for the single determinant on antigen.Therefore, modifier " monoclonal " indicates the feature of this antibody to be that basically homogeneous antibody population obtains, and shall not be construed as and require to produce this antibody by any ad hoc approach.Such as, can be produced by multiple technologies and treat monoclonal antibody used according to the invention, described technology includes but not limited to that hybridoma method, recombinant DNA method, phage display method and utilization contain the method for the transgenic animal of all or part of human immunoglobulin gene's seat, there is described herein these class methods for generation of monoclonal antibody and other illustrative methods.

" natural antibody " refers to the naturally occurring immunoglobulin molecules with different structure.Such as, about 150, the 000 daltonian different tetramer glycoprotein that is made up of two identical light chains heavy chain identical with two of disulfide-bonded of native IgG antibodies.From N end to C end, every bar heavy chain has variable region (VH), also referred to as Weight variable structural domain or heavy-chain variable domains, is three constant domain (CH1, CH2 and CH3) subsequently.Similarly, from N end to C end, every bar light chain has variable region (VL), also referred to as variable light structure territory or light variable domains, is constant light (CL) structural domain subsequently.The light chain of antibody can be divided into one of two types based on the aminoacid sequence of its constant domain, is called κ (κ) and λ (λ).

Term " package insert " is used to refer to the habitual specification sheets comprised in the commercial package for the treatment of product, and described specification sheets contains about indication, usage, dosage, uses, conjoint therapy, contraindication and/or relate to the information of warning of this type for the treatment of end-use.

Relative to reference polypeptide sequence, " amino acid sequence identity percentage ratio (%) " is defined as aligned sequences and as required introduce room using realize maximal sequence percent identity and do not consider any preservative replacement as sequence iden part after, the percentage ratio of amino-acid residue identical with the amino-acid residue in reference polypeptide sequence in candidate sequence.In order to determine that the comparison of amino acid sequence identity percentage ratio can realize by the various ways in the limit of power of this area, such as, use the computer software that can openly obtain as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the suitable parameter of aligned sequences, comprise for realizing within the scope of the full length sequence that comparing high specific to required any algorithm.But, for this paper object, use gene comparision computer program ALIGN-2 to produce amino acid sequence identity % value.ALIGN-2 gene comparision computer program is authorized by Genentech, Inc., and source code has been committed to the U.S. Copyright Office of Washington D.C. 20559 with customer documentation, and it is registered with S. Copyright registration number TXU510087 there.ALIGN-2 program is from Genentech, Inc., SouthSanFrancisco, and California openly can obtain or can collect from source code.Should by ALIGN-2 program assembly in the upper use of UNIX operating system (comprising digital UNIXV4.0D).Full sequence compares parameter by ALIGN-2 program setting and does not change.

When using ALIGN-2 comparing amino acid sequence, calculate given aminoacid sequence A and given aminoacid sequence B as follows, with given aminoacid sequence B or the amino acid sequence identity % for given aminoacid sequence B (this can alternatively be described as with given aminoacid sequence B, with given aminoacid sequence B or the given aminoacid sequence A for given aminoacid sequence B or comprise with certain amino acid sequence identity %):

100 are multiplied by mark X/Y

X be by A and the B comparison result of alignment programs ALIGN-2 in this program in be assessed as the number of the amino-acid residue of identical match, and wherein Y is the sum of amino-acid residue in B.It will be appreciated that when the length of aminoacid sequence A is not equal to the length of aminoacid sequence B, A will be not equal to the amino acid sequence identity % of B relative to A relative to the amino acid sequence identity % of B.Unless expressly stated otherwise, otherwise whole amino acid sequence identity value % used herein uses ALIGN-2 computer program to obtain as described in immediately leading portion.

Term " pharmaceutical preparation " refers to a kind of prepared product, and described prepared product is in this kind of form thus allows the biological activity of wherein contained activeconstituents effective, and containing for said preparation by additional component unacceptably poisonous for the object that is applied to.

" pharmaceutically acceptable carrier " to refer in pharmaceutical preparation the composition nontoxic to object in addition to the active ingredient (s).Pharmaceutically acceptable carrier includes but not limited to buffer reagent, vehicle, stablizer or sanitas.

" peptide linker " refers to the peptide with aminoacid sequence as the term is employed herein, and described peptide is synthesis source in one embodiment.Peptide linker is the peptide of the aminoacid sequence with at least 30 amino acid lengths in one embodiment, is the peptide of the aminoacid sequence with 32 to 50 amino acid lengths in one embodiment.In one embodiment, peptide linker is the peptide of the aminoacid sequence with 32 to 40 amino acid lengths.In one embodiment, peptide linker is (GxS) n, wherein G=glycine, S=Serine, (x=3, n=8,9 or 10) or (x=4 and n=6,7 or 8), in one embodiment, x=4, n=6 or 7, in one embodiment wherein: x=4, n=7.In one embodiment, peptide linker is (G4S) ₆g ₂.

Term " recombinant antibodies " refers to by recombinant means preparation, whole antibody (chimeric, humanization and people's antibody) of expressing, produce or being separated.This comprise from host cell as NS0 or Chinese hamster ovary celI or from for human immunoglobulin gene for the antibody that is separated genetically modified animal (such as mouse) or use antibody expressed by transfection to the recombinant expression vector in host cell.This type of recombinant antibodies has variable region and the constant region of rearranged form.As recombinant antibodies reported here can experience somatic hypermutation in body.Therefore, although the aminoacid sequence in VH and the VL district of recombinant antibodies is derived from people germline VH and VL sequence and associated, the sequence of inside, people's antibody germline storehouse in body can not be naturally present in.

As used herein, noun " treatment " (with its grammatical variants as " treatment " or " treatment ") refers to the clinical intervention being intended to change the natural process connecing subject individuality, and can be intended to prevention or implement during clinical pathology process.The result for the treatment of wanted comprises, but be not limited to, prevent disease from occurring or recurrence, mitigation symptoms, reduction disease any direct or indirect pathological consequences, prevent transfer, reduce disease progression speed, improvement or relax morbid state, and to alleviate or prognosis improvement.In some embodiments, the formation or be used for being used for delaying disease as antibody reported here or Fc district fusion polypeptide is slowed down the progress of disease.

As used in this application, term " valence state " refers to there is the binding site specified number in (antibody) molecule.So, term " bivalent ", " tetravalence " and " sexavalence " refer to there are 2 binding sites, 4 binding sites and 6 binding sites in (antibody) molecule respectively.In a preferred embodiment, the bi-specific antibody as reported here is " bivalent ".

Term " variable region " or " variable domains " refer to the structural domain of its antigen of participation antibodies of heavy chain of antibody or light chain.Heavy chain of antibody has similar structure usually with the variable domains (being VH with VL respectively) of light chain, often kind of structural domain comprise four framework regions (FR) and three hypervariable regions (HVR) (see, such as Kindt, T.J. people is waited, KubyImmunology, the 6th edition, W.H.FreemanandCo., N.Y. (2007), the 91st page).Single VH or VL structural domain may be enough to give antigen-binding specificity.In addition, the antibody in conjunction with specific antigen can utilize VH or the VL structural domain from the antibody of conjugated antigen to carry out the library being separated to screen complementary VL or VH structural domain respectively.See, such as, the people such as Portolano, S., J.Immunol.150 (1993) 880-887; The people such as Clackson, T., Nature352 (1991) 624-628).

Term " Ocular Vessels disease " includes but not limited to that intraocular neovascularization syndrome is as diabetic retinopathy, diabetic macular edema, retinopathy of prematurity, neovascular glaucoma, retinal vein occlusion, central retinal vein occlusion, macular degeneration, age-related macular degeneration, retinitis pigmentosa, retinal angiomatous is bred, macula lutea telangiectasia, ischemic retinopathy, iris neovascularization, intraocular neovascularization, corneal vascularization, retinal neovascularization, choroidal neovascularization and retinal degeneration are (see such as Garner, A., Vasculardiseases, draw certainly: Pathobiologyofoculardisease, Adynamicapproach, Garner, and Klintworth A., G.K., (writing), 2nd edition, MarcelDekker, NewYork (1994), 1625-1710 page).

As used herein, term " carrier " refers to the nucleic acid molecule can breeding connected another kind of nucleic acid.This term comprise as self-replacation type nucleic acid construct carrier and be incorporated to and this carrier introduced the carrier in the genome of host cell wherein.Some carrier can instruct the expression of nucleic acid be effectively connected with them.Examples of such carriers is called in this article " expression vector ".

" VEGF " refers to human vascular endothelial growth factor (VEGF/VEGF-A) as the term is employed herein, 165 amino acid human vascular endothelial growth factors (the amino acid 27-191:SEQIDNO:30 of the precursor sequence of people VEGF165; Amino acid/11-26 representation signal peptide), and 121,189 and 206 relevant vascular endothelial growth factor isotypes, as people such as Leung, D.W., Science246 (1989) 1306-1309; The people such as Houck, Mol.Endocrin.5 (1991) 1806-1814; The people such as Keck, P.J., the people such as Science246 (1989) 1309-1312 and Connolly, D.T., described by J.Biol.Chem.264 (1989) 20017-20024; Together with naturally occurring allelotrope and the form processing of these somatomedins.VEGF participates in regulating normal and abnormal vascular generation and the neovascularization relevant to tumour and intra-ocular disorders (people such as Ferrara, N., Endocrin.Rev.18 (1997) 4-25; The people such as Berkman, R.A., J.Clin.Invest.91 (1993) 153-159; The people such as Brown, L.F., HumanPathol.26 (1995) 86-91; The people such as Brown, L.F., CancerRes.53 (1993) 4727-4735; The people such as Mattern, J., Brit.J.Cancer.73 (1996) 931-934; And the people such as Dvorak, H.F., Am.J.Pathol.146 (1995) 1029-1039).VEGF has been separated from several source and has comprised the glycoprotein of the homodimeric of several isotype.The mitogenic activity of VEGF Human Umbilical Vein Endothelial Cells display high special.

" there is sudden change IHH-AAA " as the term is employed herein and refer to suddenly change in the constant heavy district of IgG1 or IgG4 subclass I253A (Ile253Ala), the combination of H310A (His310Ala) and H435A (His435Ala), and " there is sudden change HHY-AAA " as the term is employed herein and refer to suddenly change in the constant heavy district of IgG1 or IgG4 subclass H310A (His310Ala), the combination of H433A (His433Ala) and Y436A (Tyr436Ala), and " there is sudden change YTE " as the term is employed herein and refer to suddenly change in the constant heavy district of IgG1 or IgG4 subclass M252Y (Met252Tyr), the combination of S254T (Ser254Thr) and T256E (Thr256Glu), wherein number the EUindex according to Kabat.

" there is sudden change P329GLALA " as the term is employed herein and refer to the combination of L234A (Leu234Ala), L235A (Leu235Ala) and P329G (Pro329Gly) of suddenling change in the constant heavy district of IgG1 subclass, wherein number the EUindex according to Kabat." there is sudden change SPLE " as the term is employed herein and refer to the combination of S228P (Ser228Pro) and L235E (Leu235Glu) of suddenling change in the constant heavy district of IgG4 subclass, wherein number the EUindex according to Kabat." have sudden change SPLE and P239G " refers to suddenly change in the constant heavy district of IgG4 subclass the combination of S228P (Ser228Pro), L235E (Leu235Glu) and P329G (Pro329Gly) as the term is employed herein, wherein numbers the EUindex according to Kabat.

II. the present invention

The present invention is at least in part based on following discovery: the FcRn keying action of antibody or Fc district fusion polypeptide can be adjusted by the amino-acid residue changed in the non-corresponding position of Ge Tiao Fc district polypeptide, and reason is that these changes jointly play a role in adjustment FcRn keying action.If antibody reported here and Fc district fusion polypeptide are useful, such as, be used for the treatment of the disease wherein requiring customization general retention time.

A. newborn Fc-acceptor (FcRn)

Newborn Fc-acceptor (FcRn) is important to the metabolic fate in IgG antibody-like body.FcRn plays the effect of the wild-type IgG remedied from lysosomal degradation pathway, causes clearance rate to reduce and transformation period increase.It is by two polypeptide: the heterodimeric protein that 50kDa major histocompatibility complex sample protein I class (α-FcRn) and 15kDa B2M (β 2m) form.FcRn combines with the CH2-CH3 in the Fc district of high-affinity and IgG antibody-like part.Interaction between IgG antibody-like and FcRn strictly relies on pH and occurs with 1:2 stoichiometry, namely IgG antibody molecule can by its two heavy chain Fc district's polypeptide and two FcRn interactions of molecules (see, such as Huber, A.H. people is waited, J.Mol.Biol.230 (1993) 1077-1083).

Therefore, the external FcRn binding characteristic/feature of IgG indicates pharmacokinetic properties in its body in blood circulation.

The different aminoacids residue of heavy chain CH2 structural domain and CH3 structural domain participates in the interaction between FcRn and IgG antibody-like Fc district.And the interactional amino-acid residue of FcRn between about EU position 243 and EU position 261, approximately between EU position 275 and EU position 293, approximately between EU position 302 and EU position 319, approximately between EU position 336 and EU position 348, approximately EU position 367 and EU position 393, at EU position 408 place and approximately between EU position 424 and EU position 440.More specifically, the interaction between Fc district and FcRn is participated according to the following amino-acid residue of KabatEU numbering: F243, P244, P245P, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, I336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F372, Y373, P374, S375, D376, I377, A378, V379, E380, W381, E382, S383, N384, G385, Q386, P387, E388, N389, Y391, T393, S408, S424, C425, S426, V427, M428, H429, E430, A431, L432, H433, N434, H435, Y436, T437, Q438, K439 and S440.

Site-directed mutagenesis research has proved that the key binding sites of FcRn in the Fc district of IgG is Histidine 310, Histidine 435 and Isoleucine 253 and more low degree is that Histidine 433 and trorsine 14 36 are (see such as Kim, J.K. people is waited, Eur.J.Immunol.29 (1999) 2819-2825; The people such as Raghavan, M., Biochem.34 (1995) 14649-14657; The people such as Medesan, C., J.Immunol.158 (1997) 2211-2217).

Increase the method for IgG and FcRn combination by implementing at following multiple amino acids residue place sudden change IgG: Threonine 250, methionine(Met) 252, Serine 254, Threonine 256, Threonine 307, L-glutamic acid 380, methionine(Met) 428, Histidine 433 and l-asparagine 434 are (see Kuo, T.T. people is waited, J.Clin.Immunol.30 (2010) 777-789).

Need the antibody that the transformation period in blood circulation reduces in some cases.Such as, the long half-lift that vitreous body oral medicine thing should having in eye and patient circulation in there is short-half-life.This antibody-like also has the advantage that disease location increases as exposed amount in eye.

It is known for affecting FcRn combination and affecting the difference sudden change of transformation period in blood circulation.Identified by site-directed mutagenesis and mouse Fc-mouse FcRn has been interacted vital Fc district residue (see such as Dall ' Acqua, the people such as W.F., J.Immunol169 (2002) 5171-5180).Residue I253, H310, H433, N434 and H435 (EU according to Kabat numbers) participate in this interaction (people such as Medesan, C., Eur.J.Immunol.26 (1996) 2533-2536; The people such as Firan, M., Int.Immunol.13 (2001) 993-1002; The people such as Kim, J.K., Eur.J.Immunol.24 (1994) 542-548)).Find the interaction of residue I253, H310 and H435 to people Fc and mouse FcRn most important people such as (, Eur.J.Immunol.29 (1999) 2819-2825) Kim, J.K..Studied by protein-protein interaction, the people such as Dall ' Acqua have described residue M252Y, S254T, T256E and have improved FcRn combination (Dall'Acqua, W.F. people is waited, J.Biol.Chem.281 (2006) 23514-23524).The research of people Fc-people FcRn mixture has shown residue I253, S254, H435 and Y436 to this interaction most important (people such as Firan, M., Int.Immunol.13 (2001) 993-1002; The people such as Shields, R.L., J.Biol.Chem.276 (2001) 6591-6604).In the people such as Yeung, Y.A. (J.Immunol.182 (2009) 7667-7671), report and have studied the various mutant of residue 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437.Exemplary mutations is listed with them to the impact of FcRn keying action in following table.

table

Have been found that a sudden change of side in a Fc district polypeptide is enough to significantly weaken the keying action with Fc acceptor.Introduce more multimutation in Fc district, then become more weak with the keying action of FcRn.But one-sided asymmetric sudden change is not enough to suppress FcRn keying action completely.Sudden change on bilateral suppresses FcRn keying action required completely.

Therefore, variant (people) IgG class Fc district be heterodimer and first (heavy chain) Fc district's polypeptide and second (heavy chain) Fc district polypeptide pairing to form the formation that functional Fc district causes heterodimer.

The symmetrical through engineering approaches showing IgG1Fc district in following table affects the result of FcRn keying action (sudden change and FcRn-affinity column on the contrast of retention time).

Table

Lower than the retention time of 3 minutes correspond to do not combine because material be in through-flow in (peak, space).

Single mutation H310A is the most reticent symmetry sudden change eliminating any FcRn keying action.

Symmetrical single mutation I253A and H435A causes retention time relativity shift 0.3-0.4 minute.This can be considered as undetectable keying action usually.

Single mutation Y436A causes interaction strength that is detectable and FcRn affinity column.When not by this theory constraint, this sudden change may have can interacting the transformation period as different in I253A, H310A and H435A mutation combination (IHH-AAA suddenlys change) from zero of FcRn mediation.

The result (about reference, see WO2006/031370) obtained with the symmetrical Anti-HER 2 modified is shown in following table.

table

In Fc district, introduce the effect affecting the asymmetric sudden change of FcRn keying action has used dual specific anti-vegf/ANG2 antibody (seeing below) to exemplify, wherein said antibody use projection-enter the assembling of-hole (knobs-into-holes) technology (see such as US7,695,936, US2003/0078385; " projection chain " suddenlys change: S354C/T366W, and " pore chain " suddenlys change: Y349C/T366S/L368A/Y407V).The sudden change of asymmetric introducing can use FcRn affinity chromatography easily to determine (see Fig. 9 and following table) on the impact of FcRn keying action.From FcRn affinity column wash-out a little later, the antibody namely on FcRn affinity column with longer retention time has longer Half-life in vivo, and vice versa.

table

Asymmetric IHH-AAA-and LLL-DDD-sudden change (LLL-DDD-sudden change=L251D, L314D and L432D) shows than corresponding parent or the more weak keying action of wild-type antibodies.

Symmetrical HHY-AAA sudden change (=mutation combination H310A, H433A and Y436A) generation is no longer combined with people FcRn, the Fc district (see Figure 11,12,13 and 14) of albumin A keying action maintenance simultaneously.

In Fc district, introduce the effect affecting the asymmetric sudden change of FcRn keying action used the anti-IGF-1R antibody (IGF-1R) of dual specific anti-vegf/ANG2 antibody (VEGF/ANG2), monospecific and exemplified further with the full length antibody (fusions) that the C-terminal of two heavy chains merges, wherein said antibody use projection-enter-hole technology assembling is to allow to introduce asymmetric sudden change (see such as US7,695,936, US2003/0078385; " projection chain " suddenlys change: S354C/T366W, and " pore chain " suddenlys change: Y349C/T366S/L368A/Y407V).The impact of sudden change on FcRn keying action and albumin A keying action of asymmetric introducing can use FcRn affinity chromatography, protein A-sepharose affinity chromatography and the method based on SPR easily to determine (seeing table).

Mutation combination I253A, H310A, H435A or L251D, L314D, L432D or L251S, L314S, L432S cause losing the keying action of albumin A, and mutation combination I253A, H310A, H435A or H310A, H433A, Y436A or L251D, L314D, L432D cause losing people's neonatal Fc receptor keying action.

If reported here aspect comprises antibody as variant human IgG class Fc district reported here or Fc district fusion polypeptide.

Fusion polypeptide Zhong Fc district of Fc district gives above-mentioned feature to its fusion partner.Fusion partner can be have bioactive any molecule, wherein should reduce or increase the Half-life in vivo of described molecule, namely should clearly limit for the expection application of described molecule or customize its Half-life in vivo.

Fc district fusion polypeptide can such as comprise as variant (people) IgG class Fc district reported here and the receptor protein that combines with target (comprising part), as, such as, TNFR-Fc district fusion polypeptide (TNFR=human tumor necrosis factor receptor) or IL-1R-Fc district fusion polypeptide (IL-1R=human interleukin-1 acceptor) or VEGFR-Fc district fusion polypeptide (VEGFR=human vascular endothelial growth factor acceptor) or ANG2R-Fc district fusion polypeptide (ANG2R=human angiogenin 2 acceptor).

Fc district fusion polypeptide can comprise such as variant (people) IgG class Fc district reported here and the antibody fragment that combines with target (comprising part), such as, Fab fragments, scFv (see such as Nat.Biotechnol.23 (2005) 1126-1136), or domain antibodies (dAb) (see such as WO2004/058821, WO2003/002609).

Fc district fusion polypeptide can such as comprise as variant (people) IgG class Fc district reported here and (natural existence or artificial) receptors ligand.

B. exemplary antibodies

In one aspect, the invention provides the separation antibody with modulated FcRn keying action, namely with do not affect FcRn keying action sudden change antibody compared with, these antibody are combined with people FcRn with higher or lower avidity.

In one embodiment, antibody comprises Fc district, and described Fc district comprises a Fc district polypeptide and the 2nd Fc district polypeptide,

Wherein

A) a Fc district polypeptide and the 2nd Fc district polypeptide are derived from identical Ren Fc district polypeptide, and

B) a Fc district polypeptide is modified, be that its aminoacid sequence is at least different from the 2nd Fc district polypeptid acid sequence in a corresponding position according to KabatEUindex number system, and the 2nd Fc district polypeptide is modified, be that its aminoacid sequence is at least different from a Fc district polypeptid acid sequence in a corresponding position according to KabatEUindex number system, thus the modification position in a Fc district polypeptide and the modification position in the 2nd Fc district polypeptide are different, and

C) with comprise a) Ren Fc district polypeptide (namely at the polypeptide according to the corresponding position of KabatEUindex number system with the amino-acid residue identical with a) Ren Fc district polypeptide) as the first and second Fc district polypeptide Fc district compared with, this Fc district has the different avidity to people Fc-acceptor.

The dual specific bivalent antibody that a kind of exemplary antibodies as reported here is and one aspect of the present invention or FcRn keying action are eliminated, described antibody comprises and the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding

Wherein

I) in heavy-chain variable domains, the CDR1H district of the CDR3H district of SEQIDNO:14, the CDR2H district of SEQIDNO:15 and SEQIDNO:16 is comprised with the first antigen binding site of VEGF specific binding, and in light variable domains, comprise the CDR1L district of the CDR3L district of SEQIDNO:17, the CDR2L district of SEQIDNO:18 and SEQIDNO:19, and

Ii) in heavy-chain variable domains, the CDR1H district of the CDR3H district of SEQIDNO:22, the CDR2H district of SEQIDNO:23 and SEQIDNO:24 is comprised with the second antigen binding site of ANG-2 specific binding, and in light variable domains, comprise the CDR1L district of the CDR3L district of SEQIDNO:25, the CDR2L district of SEQIDNO:26 and SEQIDNO:27, and

Iii) bi-specific antibody comprises Fc district, and described Fc district comprises a Fc district polypeptide and the 2nd Fc district polypeptide,

Wherein

In an embodiment in whole, Fc district is variant (people) IgG class Fc district.In one embodiment, variant (people) IgG class Fc district is the different dimerization Fc district of IgG class.

In an embodiment in whole, the formation of a Fc district polypeptide and the polypeptide pairing of the 2nd Fc district (have function) being formed Fc district causes heterodimer.

In one embodiment, people Fc district polypeptide is IgG1 subclass or IgG4 subclass Ren Fc district polypeptide.

In one embodiment, people Fc district polypeptide is IgG1 subclass Ren Fc district polypeptide, and it also comprises sudden change L234A, L235A and P329G.

In one embodiment, people Fc district polypeptide is IgG4 subclass Ren Fc district polypeptide, and it also comprises sudden change S228P and L235E.

In one embodiment, a Fc district polypeptide also comprise sudden change S354C and T366W and the 2nd Fc district polypeptide also comprise sudden change Y349C, T366S, L368A and Y407V.

In one embodiment, bi-specific antibody is characterised in that

I) aminoacid sequence comprising SEQIDNO:20 with the first antigen binding site of VEGF specific binding as heavy-chain variable domains VH and the aminoacid sequence comprising SEQIDNO:21 as light variable domains VL, and

Ii) aminoacid sequence comprising SEQIDNO:28 with the second antigen binding site of ANG-2 specific binding as heavy-chain variable domains VH and the aminoacid sequence comprising SEQIDNO:29 as light variable domains VL.

In one embodiment, the feature of bi-specific antibody is iii) Fc district belong to human IgG1's subclass.In one embodiment, the feature of bi-specific antibody is that the Fc district of human IgG1's subclass also comprises sudden change L234A, L235A and P329G (EUindex according to Kabat numbers).

In one embodiment, the feature of bi-specific antibody is iii) Fc district belong to human IgG 4 subclass.In one embodiment, the feature of bi-specific antibody is that the Fc district of human IgG 4 subclass also comprises sudden change S228P and L235E (EUindex according to Kabat numbers).In one embodiment, the feature of bi-specific antibody is that the Fc district of human IgG 4 subclass also comprises sudden change S228P, L235E and P329G (EUindex according to Kabat numbers).

In one embodiment, bi-specific antibody comprises Fc district, and described Fc district comprises the first and second Fc district polypeptide of human IgG1 or human IgG 4 subclass (i.e. derived from human source), and described polypeptide comprises to be selected from a Fc district polypeptide i) organizes I253A, H310A, H435A, or ii) organize H310A, H433A, Y436A, or iii) organize L251D, L314D, L432D, or iv) organize L251S, L314S, L432S (according to KabatEUindex number system numbering) one or two sudden change and comprise in the 2nd Fc district polypeptide be selected from sudden change L251D, L251S, I253A, H310A, L314D, L314S, L432D, L432S, H433A, H435A, one or two sudden change of Y436A (according to KabatEUindex number system numbering), thus the i) I253A that all suddenlys change, H310A, H435A, or ii) H310A, H433A, Y436A, or iii) L251D, L314D, L432D, or iv) L251S, L314S, L432S is all contained in this Fc district.

In one embodiment, bi-specific antibody comprises Fc district, described Fc district comprises the first and second Fc district polypeptide of human IgG1 or human IgG 4 subclass (derived from human source), described polypeptide comprises sudden change I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D or L251S/L314S/L432S or its combination (according to KabatEUindex number system numbering) in Fc district, thus all sudden change all first or the 2nd in Fc district polypeptide, or one or two sudden change in a Fc district polypeptide and one or two sudden change in the 2nd Fc district polypeptide, thus the i) I253A that all suddenlys change, H310A, H435A, or ii) H310A, H433A, Y436A, or iii) L251D, L314D, L432D, or iv) L251S, L314S and L432S is all contained in this Fc district.

Still as other aspects reported here be comprise the pharmaceutical preparation of bi-specific antibody, purposes that the pharmaceutical preparation being used for the treatment of Ocular Vessels disease, bi-specific antibody manufacture are used for the treatment of the medicine of Ocular Vessels disease, by using bi-specific antibody to the method needing the patient treatment of this treatment to suffer from the patient of Ocular Vessels disease.In one embodiment, use bi-specific antibody by application in vitreum or comprise the pharmaceutical preparation of bi-specific antibody.

Another aspect of the present invention is that coding is as the heavy chain of bi-specific antibody reported here and/or the nucleic acid molecule of light chain.

The present invention also provides the expression vector (described expression vector can express this nucleic acid in protokaryon or eukaryotic host cell) containing, for example nucleic acid reported here, and containing this kind of carrier for the host cell of generation as bi-specific antibody reported here of recombinating.

The present invention also comprises protokaryon or eukaryotic host cell, and described host cell comprises as carrier reported here.

The present invention also comprises a kind of method for generation of bi-specific antibody such as reported here, and the feature of described method is as follows: express in protokaryon or eukaryotic host cell as nucleic acid reported here and reclaim bi-specific antibody from cell or cell culture supernatant.An embodiment is a kind of method for the preparation of bi-specific antibody such as reported here, and described method comprises step

A) with the vector host cell of nucleic acid molecule comprising encoding antibody;

B) under the condition allowing synthetic antibody, host cell is cultivated; And

C) antibody is reclaimed from culture.

The present invention also comprises the antibody obtained by the method for this generation bi-specific antibody.

Have highly valuable characteristic as the specific modification in antibody Yin Qi Fc district reported here, described modification is to the benefits subjects place suffering from Ocular Vessels disease.They show high stability and slowly spread (compared with the less antibody fragment without constant heavy district) from eye in vitreum environment, and wherein actual disease exists and is treated (therefore with non-IgG sample antibody such as Fab fragment and (Fab) ₂fragment is compared, and treatment plan can improve potentially).On the other hand, as antibody reported here rapidly clear from serum (this be in the urgent need to reduce the potential side effect being derived from systemic exposure).Surprisingly, they also show comparatively low viscosity (see Fig. 2) (with in constant region without mutation combination I253A, the form of H310A with H435A is compared) and be therefore be used for the treatment of illness in eye especially during carry out applying in vitreum (for this kind of application, general use fine needle and high viscosity makes the applying quite difficulty that is suitable for) by fine needle.The preparation of greater concn is also allowed compared with low viscosity.

Also surprisingly, antibody as reported here be presented at gathering proneness (see Fig. 4) lower between the shelf lives (with in Fc district without mutation combination I253A, the form of H310A with H435A is compared), this is to applying in vitreum in eye most important (because the gathering in eye may cause complication at this kind of treatments period).

As bi-specific antibody reported here shows good effect in suppression vascular disease.

In certain embodiments, as bi-specific antibody reported here shows valuable characteristic because of its specific modification (such as P329GLALA) in constant region, if not being bonded to Fc γ acceptor/without the effect of Fc γ receptors bind, this reduces the risk of side effect as thrombosis and/or undesirable necrocytosis (caused by such as ADCC).

In an embodiment such as reported here, the bi-specific antibody as reported here is bivalent.

In one aspect of the invention, the feature as dual specific bivalent antibody reported here is to comprise

A) with heavy chain and the light chain of the first full length antibody of VEGF specific binding, and

B) with the modification heavy chain of the second full length antibody of ANG-2 specific binding with modify light chain, wherein constant domain CL and CH1 replaces mutually.

This with human vascular endothelial growth factor (VEGF) and the dual specific bivalent antibody pattern of the bi-specific antibody of human angiopoietin-2 (ANG-2) specific binding describe in WO2009/080253 (comprising the CH3 structural domain that projection-enter-hole is modified).Based on the antibody called after CrossMab of this dual specific bivalent antibody pattern.

In one embodiment, the feature of this kind of dual specific bivalent antibody is to comprise

A) aminoacid sequence of SEQIDNO:38 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:40 light chain as the first full length antibody, and

B) aminoacid sequence of SEQIDNO:39 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:41 modification light chain as the second full length antibody.

A) aminoacid sequence of SEQIDNO:34 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:36 light chain as the first full length antibody, and

B) aminoacid sequence of SEQIDNO:35 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:37 modification light chain as the second full length antibody.

A) aminoacid sequence of SEQIDNO:42 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:44 light chain as the first full length antibody, and

B) aminoacid sequence of SEQIDNO:43 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:45 modification light chain as the second full length antibody.

A) aminoacid sequence of SEQIDNO:90 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:40 light chain as the first full length antibody, and

B) aminoacid sequence of SEQIDNO:91 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:41 modification light chain as the second full length antibody.

A) aminoacid sequence of SEQIDNO:88 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:36 light chain as the first full length antibody, and

B) aminoacid sequence of SEQIDNO:89 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:37 modification light chain as the second full length antibody.

A) aminoacid sequence of SEQIDNO:92 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:44 light chain as the first full length antibody, and

B) aminoacid sequence of SEQIDNO:93 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:45 modification light chain as the second full length antibody.

Therefore if an aspect reported here is a kind of dual specific bivalent antibody comprised with the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, it is characterized in that the aminoacid sequence comprising SEQIDNO:38, SEQIDNO:39, SEQIDNO:40 and SEQIDNO:41.

Therefore if an aspect reported here is a kind of dual specific bivalent antibody comprised with the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, it is characterized in that the aminoacid sequence comprising SEQIDNO:34, SEQIDNO:35, SEQIDNO:36 and SEQIDNO:37.

Therefore if an aspect reported here is a kind of dual specific bivalent antibody comprised with the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, it is characterized in that the aminoacid sequence comprising SEQIDNO:42, SEQIDNO:43, SEQIDNO:44 and SEQIDNO:45.

Therefore if an aspect reported here is a kind of dual specific bivalent antibody comprised with the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, it is characterized in that the aminoacid sequence comprising SEQIDNO:90, SEQIDNO:91, SEQIDNO:40 and SEQIDNO:41.

Therefore if an aspect reported here is a kind of dual specific bivalent antibody comprised with the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, it is characterized in that the aminoacid sequence comprising SEQIDNO:88, SEQIDNO:89, SEQIDNO:36 and SEQIDNO:37.

Therefore if an aspect reported here is a kind of dual specific bivalent antibody comprised with the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, it is characterized in that the aminoacid sequence comprising SEQIDNO:92, SEQIDNO:93, SEQIDNO:44 and SEQIDNO:45.

In such as reported here another, the feature as bi-specific antibody reported here is to comprise

B) with heavy chain and the light chain of the second full length antibody of ANG-2 specific binding, wherein the N-terminal of heavy chain is connected with the C-terminal of light chain by peptide linker.

This with human vascular endothelial growth factor (VEGF) and the dual specific bivalent antibody pattern of the bi-specific antibody of human angiopoietin-2 (ANG-2) specific binding describe in WO2011/117330 (comprising the CH3 structural domain that projection-enter-hole is modified).Based on antibody called after single armed strand pattern Fab (OAscFab) of this dual specific bivalent antibody pattern.

A) aminoacid sequence of SEQIDNO:46 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:48 light chain as the first full length antibody, and

B) aminoacid sequence of SEQIDNO:47 is as the heavy chain of the second full length antibody be connected with the light chain of the second full length antibody by peptide linker.

A) aminoacid sequence of SEQIDNO:49 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:51 light chain as the first full length antibody, and

B) aminoacid sequence of SEQIDNO:50 is as the heavy chain of the second full length antibody be connected with the light chain of the second full length antibody by peptide linker.

A) aminoacid sequence of SEQIDNO:94 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:48 light chain as the first full length antibody, and

B) aminoacid sequence of SEQIDNO:95 is as the heavy chain of the second full length antibody be connected with the light chain of the second full length antibody by peptide linker.

A) aminoacid sequence of SEQIDNO:96 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:51 light chain as the first full length antibody, and

B) aminoacid sequence of SEQIDNO:97 is as the heavy chain of the second full length antibody be connected with the light chain of the second full length antibody by peptide linker.

In one embodiment, by introducing the heavy chain of disulfide linkage further stabilization second full length antibody and the heavy chain of antibody variable domains (VH) of light chain and light chain of antibody variable domains (VL) between with upper/lower positions: (Kabat (is numbered according to Kabat in heavy-chain variable domains position 44 and light variable domains position 100, E.A. people is waited, SequencesofProteinsofImmunologicalInterest, 5th edition, PublicHealthService, NationalInstitutesofHealth, Bethesda, MD (1991)).Introduce disulfide linkage to play the technology of stabilization such as at WO94/029350; The people such as Rajagopal, V., Prot.Eng.10 (1997) 1453-1459; The people such as Kobayashi, the people such as NuclearMedicine & Biology25 (1998) 387-393 and Schmidt, M., describe in Oncogene18 (1999) 1711-1721.

Therefore if an aspect reported here is a kind of dual specific bivalent antibody comprised with the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, it is characterized in that the aminoacid sequence comprising SEQIDNO:46, SEQIDNO:47 and SEQIDNO:48.

Therefore if an aspect reported here is a kind of dual specific bivalent antibody comprised with the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, it is characterized in that the aminoacid sequence comprising SEQIDNO:49, SEQIDNO:50 and SEQIDNO:51.

Therefore if an aspect reported here is a kind of dual specific bivalent antibody comprised with the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, it is characterized in that the aminoacid sequence comprising SEQIDNO:94, SEQIDNO:95 and SEQIDNO:48.

Therefore if an aspect reported here is a kind of dual specific bivalent antibody comprised with the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, it is characterized in that the aminoacid sequence comprising SEQIDNO:96, SEQIDNO:97 and SEQIDNO:51.

In one embodiment, the CH3 structural domain as dual specific bivalent antibody reported here is changed by " projection-enter-hole (knob-into-hole) " technology, described technology is people such as such as WO96/027011, RidgwayJ.B., ProteinEng.9 (1996) 617-621 and Merchant, A.M. people is waited, the several example in detail in Nat.Biotechnol.16 (1998) 677-681.In this approach, the interaction face of two CH3 structural domains is changed to increase the different dimerization of two heavy chains containing these two CH3 structural domains.The one of (two heavy chain) two CH3 structural domains can be " projection ", and another one is " hole ".Heterodimer (people such as Merchant, A.M, NatureBiotech.16 (1998) 677-681 are stablized in the introducing of disulfide linkage further; The people such as Atwell, S., J.Mol.Biol.270 (1997) 26-35) and increase productive rate.

Such as reported here whole in a preferred embodiment in, the feature of bi-specific antibody is

Article one, join in the interface that the CH3 structural domain of heavy chain and each leisure of CH3 structural domain of another heavy chain comprise initial interface between antibody CH3 structural domain,

Wherein change this interface to promote the formation of bi-specific antibody, wherein said change is characterised in that

A) the CH3 structural domain of a heavy chain is so changed,

Thus at bi-specific antibody the CH3 structural domain of an inner heavy chain of joining with the initial interface of the CH3 structural domain of another heavy chain initial interface in,

Amino-acid residue is replaced with the amino-acid residue with more bulky side chain volume, thus produce protuberance in the interface internal of the CH3 structural domain of a heavy chain, described protuberance can be placed in the chamber of the interface internal of the CH3 structural domain of another heavy chain,

And

B) the CH3 structural domain of another heavy chain is so changed,

Thus inner at bi-specific antibody, in the initial interface of the 2nd CH3 structural domain of joining with the initial interface of a CH3 structural domain,

Amino-acid residue is replaced with the amino-acid residue with less side-chain bulk, thus produce chamber in the interface internal of the 2nd CH3 structural domain, the protuberance in the interface of a CH3 structural domain can be placed in inside, described chamber.

Therefore, antibody of the present invention is characterised in that in a preferred embodiment

Join in change interface between the CH3 structural domain that each leisure of CH3 structural domain of the heavy chain of the CH3 structural domain of the heavy chain of full length antibody a) and full length antibody b) comprises antibody in initial interface,

Wherein

I) in the CH3 structural domain of a heavy chain

And wherein

Ii) in the CH3 structural domain of another heavy chain

Amino-acid residue is replaced with the amino-acid residue with less side-chain bulk, thus produce cave, chamber in the interface internal of the 2nd CH3 structural domain, described chamber can be placed at the interface internal protuberance of a CH3 structural domain inner.

In a preferred embodiment, the amino-acid residue with more bulky side chain volume is selected from arginine (R), phenylalanine (F), tyrosine (Y), tryptophane (W).

In a preferred embodiment, the amino-acid residue with less side-chain bulk is selected from L-Ala (A), Serine (S), Threonine (T), α-amino-isovaleric acid (V).

In one embodiment, two CH3 structural domains are changed further in the following manner: so in the corresponding position of each CH3 structural domain, introduce halfcystine (C) residue, thus the disulfide linkage between two CH3 structural domains can be formed.

In one embodiment, bi-specific antibody comprises T366W sudden change and in the CH3 structural domain of " pore chain ", comprises T366S, L368A and Y407V sudden change in the CH3 structural domain of " projection chain ".Also the extra interchain disulfide bond (Merchant between CH3 structural domain can be used, the people such as A.M, NatureBiotech16 (1998) 677-681), such as by " raised chain " CH3 structural domain introduce Y349C or S354C sudden change and to " pore chain " CH3 structural domain introduce Y439C or E356C or S354C sudden change.

In one embodiment, suddenly change Y349C or S354C and mutation T 366W as bi-specific antibody reported here comprises in one of two CH3 structural domains and in another structural domain of two CH3 structural domains, comprise sudden change S354C or E356C or Y349C and mutation T 366S, L368A and Y407V.In a preferred embodiment, bi-specific antibody comprises Y349C in one of two CH3 structural domains, T366W suddenlys change and comprise S354C in another structural domain of two CH3 structural domains, T366S, L368A, (EUindex according to Kabat numbers (Kabat in Y407V sudden change (the extra Y349C sudden change in a CH3 territory becomes interchain disulfide bond with the extra S354C mutant form in another CH3 territory), E.A. people is waited, SequencesofProteinsofImmunologicalInterest, 5th edition, PublicHealthService, NationalInstitutesofHealth, Bethesda, MD (1991)).In a preferred embodiment, bi-specific antibody comprises S354C in one of two CH3 structural domains, T366W suddenlys change and comprise Y349C in another structural domain of two CH3 structural domains, T366S, L368A, (EUindex according to Kabat numbers (Kabat in Y407V sudden change (the extra S354C sudden change in a CH3 territory becomes interchain disulfide bond with the extra Y349C mutant form in another CH3 territory), E.A. people is waited, SequencesofProteinsofImmunologicalInterest, 5th edition, PublicHealthService, NationalInstitutesofHealth, Bethesda, MD (1991)).

Other projection described by EP1870459A1-enter-hole technology can also be used alternative or extraly.Therefore, another example of bi-specific antibody is R409D and K370E sudden change in the CH3 structural domain of " projection chain " and D399K and E357K sudden change in the CH3 structural domain of " pore chain ") (EUindex according to Kabat numbers (Kabat, E.A. people is waited, SequencesofProteinsofImmunologicalInterest, 5th edition, PublicHealthService, NationalInstitutesofHealth, Bethesda, MD (1991)).

In another embodiment, bi-specific antibody comprises T366W sudden change and in the CH3 structural domain of " pore chain ", comprises T366S, L368A and Y407V sudden change and in the CH3 structural domain of " projection chain ", comprise R409D, K370E sudden change extraly and in the CH3 structural domain of " pore chain ", comprise D399K, E357K sudden change in the CH3 structural domain of " projection chain ".

In one embodiment, bi-specific antibody comprises Y349C in one of two CH3 structural domains, T366W suddenlys change and comprise S354C in another structural domain of two CH3 structural domains, T366S, L368A and Y407V suddenlys change, or bi-specific antibody comprises Y349C in one of two CH3 structural domains, T366W suddenlys change and comprise S354C in another structural domain of two CH3 structural domains, T366S, L368A and Y407V suddenlys change and in the CH3 structural domain of " projection chain ", comprises R409D extraly, K370E suddenlys change and comprise D399K in the CH3 structural domain of " pore chain ", E357K suddenlys change.

In one embodiment, bi-specific antibody comprises S354C in one of two CH3 structural domains, T366W suddenlys change and comprise Y349C in another structural domain of two CH3 structural domains, T366S, L368A, Y407V suddenlys change, or bi-specific antibody comprises S354C in one of two CH3 structural domains, T366W suddenlys change and comprise Y349C in another structural domain of two CH3 structural domains, T366S, L368A and Y407V suddenlys change and in the CH3 structural domain of " projection chain ", comprises R409D extraly, K370E suddenlys change and comprise D399K in the CH3 structural domain of " pore chain ", E357K suddenlys change.

In one embodiment, the bi-specific antibody as reported here is characterised in that to have following one or more characteristic:

-with without iii) under described sudden change corresponding bi-specific antibody compared with, show comparatively low-serum-concentration (be in hemizygote genetically modified mouse vitreum in apply latter 96 hour in mouse FcRn defect but to people FcRn) (measuring in assay method as described in example 6 above)

-with without iii) under described sudden change corresponding bi-specific antibody compared with, the concentration showing similar (multiple 0.8 to 1.2) in full right eye lysate (is in the genetically modified mouse of hemizygote in mouse FcRn defect but to people FcRn, latter 96 hours are applied in vitreum in right eye) (measuring in assay method as described in example 6 above)

-display is not combined with people's neonatal Fc receptor.

In one embodiment, the feature of dual specific bivalent antibody is to comprise a Fc district polypeptide and the 2nd Fc district polypeptide, wherein

A) the first and second Fc district polypeptide comprise sudden change Y436A, or

B) the first and second Fc district polypeptide comprise sudden change I253A, H310A and H435A, or

C) the first and second Fc district polypeptide comprise sudden change H310A, H433A and Y436A, or

D) the first and second Fc district polypeptide comprise sudden change L251D, L314D and L432D, or

E) the first and second Fc district polypeptide comprise sudden change L251S, L314S and L432S, or

F) a Fc district polypeptide comprise sudden change Y436A and the 2nd Fc district polypeptide comprise

-sudden change I253A, H310A and H435A, or

-sudden change H310A, H433A and Y436A, or

-sudden change L251D, L314D and L432D, or

-sudden change L251S, L314S and L432S,

Or

G) a Fc district polypeptide comprise sudden change I253A, H310A and H435A and the 2nd Fc district polypeptide comprise

-sudden change H310A, H433A and Y436A, or

-sudden change L251D, L314D and L432D, or

-sudden change L251S, L314S and L432S,

Or

H) a Fc district polypeptide comprise sudden change H310A, H433A and Y436A and the 2nd Fc district polypeptide comprise

A) suddenly change L251D, L314D and L432D, or

B) suddenly change L251S, L314S and L432S,

Or

I) a Fc district polypeptide comprise sudden change L251D, L314D and L432D and the 2nd Fc district polypeptide comprise

A) suddenly change L251S, L314S and L432S.

In one embodiment, bi-specific antibody comprises Fc district, and described Fc district comprises the first and second Fc district polypeptide of human IgG1 or human IgG 4 subclass (i.e. derived from human source), and described polypeptide comprises to be selected from a Fc district polypeptide i) organizes I253A, H310A, H435A, or ii) organize H310A, H433A, Y436A, or iii) organize L251D, L314D, L432D, or iv) organize L251S, L314S, L432S (according to KabatEUindex number system numbering) one or two sudden change and comprise in the 2nd Fc district polypeptide be selected from sudden change L251D, L251S, I253A, H310A, L314D, L314S, L432D, L432S, H433A, H435A, one or two sudden change of Y436A (according to KabatEUindex number system numbering), thus the whole sudden changes when considering in the lump in the first and second Fc district polypeptide cause sudden change i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S is contained in this Fc district.

In one embodiment, bi-specific antibody comprises Fc district, described Fc district comprises the first and second Fc district polypeptide of human IgG1 or human IgG 4 subclass (derived from human source), described polypeptide comprises sudden change I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D or L251S/L314S/L432S or its combination (according to KabatEUindex number system numbering) in Fc district, thus all sudden change all first or the 2nd in Fc district polypeptide, or one or two sudden change in a Fc district polypeptide and one or two sudden change in the 2nd Fc district polypeptide, thus the whole sudden changes when considering in the lump in the first and second Fc district polypeptide cause sudden change i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S is contained in this Fc district.

In one embodiment, the feature of dual specific bivalent antibody is to comprise

With the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, be characterised in that

I) the first antigen binding site comprise SEQIDNO:20 aminoacid sequence as heavy-chain variable domains (VH) and the aminoacid sequence comprising SEQIDNO:21 as light variable domains (VL), and

Ii) the second antigen binding site comprise SEQIDNO:28 aminoacid sequence as heavy-chain variable domains (VH) and the aminoacid sequence comprising SEQIDNO:29 as light variable domains (VL), and

Iii) bi-specific antibody comprises Fc district, and described Fc district comprises the first and second Fc district polypeptide of human IgG1 or human IgG 4 subclass (i.e. derived from human source), and described polypeptide comprises to be selected from a Fc district polypeptide i) organizes I253A, H310A, H435A, or ii) organize H310A, H433A, Y436A, or iii) organize L251D, L314D, L432D, or iv) organize L251S, L314S, L432S (according to KabatEUindex number system numbering) one or two sudden change and comprise in the 2nd Fc district polypeptide be selected from sudden change L251D, L251S, I253A, H310A, L314D, L314S, L432D, L432S, H433A, H435A, one or two sudden change of Y436A (according to KabatEUindex number system numbering), thus the whole sudden changes when considering in the lump in the first and second Fc district polypeptide cause sudden change i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S is contained in this Fc district,

And there is following one or more characteristic

-with without iii) under described sudden change corresponding bi-specific antibody compared with, show in full right eye lysate concentration (be in hemizygote genetically modified mouse vitreum in apply latter 96 hour in mouse FcRn defect but to people FcRn) (the measuring in assay method as described in example 6 above) of similar (multiple 0.8 to 1.2)

-display is not combined with people's neonatal Fc receptor.

Iii) bi-specific antibody comprises a Fc district polypeptide and the 2nd Fc district polypeptide, wherein

-sudden change I253A, H310A and H435A, or

-sudden change H310A, H433A and Y436A, or

-sudden change L251D, L314D and L432D, or

-sudden change L251S, L314S and L432S,

Or

-sudden change H310A, H433A and Y436A, or

-sudden change L251D, L314D and L432D, or

-sudden change L251S, L314S and L432S,

Or

A) suddenly change L251D, L314D and L432D, or

B) suddenly change L251S, L314S and L432S,

Or

A) suddenly change L251S, L314S and L432S,

And there is following one or more characteristic

-with without iii) under described sudden change corresponding bi-specific antibody compared with, show in full right eye lysate concentration (be in hemizygote genetically modified mouse right eye vitreum in apply latter 96 hour in mouse FcRn defect but to people FcRn) (the measuring in assay method as described in example 6 above) of similar (multiple 0.8 to 1.2)

-display is not combined with people's neonatal Fc receptor.

In one embodiment, the feature of bi-specific antibody is to comprise and the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding, is characterised in that

I) the first antigen binding site include the SEQIDNO:20 of 1,2 or 3 amino-acid substitution aminoacid sequence as heavy-chain variable domains (VH) and the aminoacid sequence including the SEQIDNO:21 of 1,2 or 3 amino-acid substitution as light variable domains (VL), and

Ii) the second antigen binding site include the SEQIDNO:28 of 1,2 or 3 amino-acid substitution aminoacid sequence as heavy-chain variable domains (VH) and the aminoacid sequence including the SEQIDNO:29 of 1,2 or 3 amino-acid substitution as light variable domains (VL), and

And there is following one or more characteristic

-display is not combined with people's neonatal Fc receptor.

Iii) bi-specific antibody comprises a Fc district polypeptide and the 2nd Fc district polypeptide wherein

-sudden change I253A, H310A and H435A, or

-sudden change H310A, H433A and Y436A, or

-sudden change L251D, L314D and L432D, or

-sudden change L251S, L314S and L432S,

Or

-sudden change H310A, H433A and Y436A, or

-sudden change L251D, L314D and L432D, or

-sudden change L251S, L314S and L432S,

Or

A) suddenly change L251D, L314D and L432D, or

B) suddenly change L251S, L314S and L432S,

Or

A) suddenly change L251S, L314S and L432S,

And there is following one or more characteristic

-display is not combined with people's neonatal Fc receptor.

Antigen binding site as bi-specific antibody reported here contains and contributes to binding site in various degree to six of the avidity of its antigen complementary determining regions (CDR).There are three heavy-chain variable domains CDR (CDRH1, CDRH2 and CDRH3) and three light variable domains CDR (CDRL1, CDRL2 and CDRL3).By comparing with the amino acid sequence database of compilation, determining the degree of CDR and framework region (FR), defining these regions according to the mutability between sequence in the database.

In an embodiment in whole, antibody not with people FcRn specific binding.In an embodiment in whole, antibody extraly with SP specific binding.

In an embodiment in whole, antibody not with people FcRn specific binding.In an embodiment in whole, antibody extraly not with SP specific binding.

In an embodiment in whole, the first polypeptide also comprises sudden change Y349C, T366S, L368A and Y407V (" hole ") and the second polypeptide comprises sudden change S354C and T366W (" projection ").

In an embodiment in whole, the first polypeptide also comprises sudden change S354C, T366S, L368A and Y407V (" hole ") and the second polypeptide comprises sudden change Y349C and T366W (" projection ").

In an embodiment in whole, Fc district polypeptide belongs to human IgG1's subclass.In one embodiment, a Fc district polypeptide and the 2nd Fc district polypeptide also comprise sudden change L234A and L235A.In one embodiment, a Fc district polypeptide and the 2nd Fc district polypeptide also comprise sudden change P329G.

In an embodiment in whole, Fc district polypeptide belongs to human IgG 4 subclass.In one embodiment, a Fc district polypeptide and the 2nd Fc district polypeptide also comprise sudden change S228P and L235E.In one embodiment, a Fc district polypeptide and the 2nd Fc district polypeptide also comprise sudden change P329G.

Antibody as reported here is produced by recombinant means.Therefore, as reported here aspect be coding as the nucleic acid of antibody reported here and another aspect is the cell comprising nucleic acid, described nucleic acid encoding is as antibody reported here.The method produced for recombinating is that this area is extensively known and be included in protein expression in prokaryotic cell prokaryocyte and eukaryotic cell, is usually purified to pharmaceutically useful purity together with later separation antibody.In order to express antibody as the aforementioned in host cell, by standard method, the nucleic acid of will encode each (modification) light chain and heavy chain inserts in expression vector.Express and carry out in suitable protokaryon or eukaryotic host cell, as Chinese hamster ovary celI, NS0 cell, SP2/0 cell, HEK293 cell, COS cell, PER.C6 cell, yeast or Bacillus coli cells and antibody reclaim from cell (cell after culture supernatant or cracking).Produce for recombinating the general method of antibody be know in prior art and such as describe in following survey article: Makrides, S.C., ProteinExpr.Purif.17 (1999) 183-202; The people such as Geisse, S., ProteinExpr.Purif.8 (1996) 271-282; Kaufman, R.J., Mol.Biotechnol.16 (2000) 151-160 and Werner, R.G., DrugRes.48 (1998) 870-880.

Therefore if an aspect reported here is a kind of method for the preparation of bi-specific antibody such as reported here, described method comprises step

A) with the vector host cell of nucleic acid molecule comprising encoding antibody,

B) under the condition allowing synthetic antibody, host cell is cultivated, and

C) antibody is reclaimed from culture.

In one embodiment, the recycling step c) comprises use light chain constant domain specificity capture agent (it is such as special to κ or λ constant light, depends on whether κ or lambda light chain are contained in bi-specific antibody).In one embodiment, this light chain specificity capture agent uses with combination-elution mode.The example of this kind of light chain constant domain specificity capture agent is such as KappaSelect ^tMand LambdaFabSelect ^tM(can obtain from GEHealthcare/BAC), it is based on the agarose base matrix of high degree of rigidity, high flow rate when described matrix allows extensive and low back pressure.These materials contain the part be combined with κ light chain or lambda light chain constant region respectively, and (constant segments namely lacking light chain will not be in conjunction with; Fig. 1).Therefore the two all can in conjunction with other target molecules containing constant region of light chain, such as, and IgG, IgA and IgM.Part connects to make them can be used for easily being combined with target molecule by wetting ability long spacer arm and matrix.They are based on the single chain antibody fragments screened for people Ig κ or λ.

In one embodiment, c) under recycling step comprise and use Fc district specificity capture agent.In one embodiment, Fc district specificity capture agent uses with combination-elution mode.The example of this kind of Fc district specificity capture agent is such as the affinity chromatographic material s based on SP.

By conventional immune globulins purification process such as, as, affinity chromatography (Protein A sepharose or KappaSelect ^tM, LambdaFabSelect ^tM), hydroxyapatite chromatography, gel electrophoresis or dialysis, bi-specific antibody is suitably separated with substratum.

Use ordinary method, DNA with RNA of encodes monoclonal antibody is separated easily and checks order.B cell or hybridoma can serve as the source of this kind of DNA and RNA.Once be separated, this DNA can be inserted expression vector, subsequently described expression vector transfection is not extremely produced in the host cell (as HEK293 cell, Chinese hamster ovary celI or myeloma cell) of immunoglobulin (Ig), to realize the synthesis of recombinant monoclonal antibodies in host cell in addition.

By comprising the Fc district of experience different modifying, some molecules as reported here provide convenient separation/purification effect, wherein at least one modify cause i) described molecule to the otherness avidity of (staphylococcus) albumin A and ii) described molecule is to the otherness avidity of people FcRn, and described molecule can be separated from the cell of fragmentation, substratum or molecule mixture the avidity of albumin A based on it.

Antibody purification is carried out by standard technique (comprising alkali/SDS process, CsCl zone method, column chromatography, agarose gel electrophoresis) and other technologies well known in the art, object is elimination cellular component or other pollutents (such as, other cellularity nucleic acid or protein) (see such as Ausubel, F. people is waited, write, CurrentProtocolsinMolecularBiology, GreenePublishingandWileyInterscience, NewYork (1987)).Fully establish diverse ways and they are widely used in protein purification, as used the affinity chromatography (such as albumin A or protein g affinity chromatography) of microbial proteinous, ion exchange chromatography (such as cationic exchange (carboxymethyl resin), anionresin (amino-ethyl resin) and mixed mode exchange), parent's sulphur adsorption (such as adopting beta-mercaptoethanol and other SH parts), hydrophobic interaction or aromatics adsorption chromatography (such as adopt Phenyl-Sepharose, parent's azepine-aromatic resins (aza-arenophilicresin) or m-aminophenyl boronic acid), metallo-chelate affinity chromatography (such as adopting Ni (II)-and Cu (II)-affinitive material), size exclusion chromatography and electrophoresis method are (as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M.A., Appl.Biochem.Biotech.75 (1998) 93-102).

The bivalent bi-specific antibody for people VEGF and people ANG-2 as reported here can possess valuable effect/safety spectrum (safetyprofile) and can for the patient benefit needing anti-vegf and anti-ANG-2 to treat.

If reported here aspect comprises the pharmaceutical preparation as antibody reported here.If reported here aspect is if antibody reported here is for the manufacture of the purposes in pharmaceutical preparation.Another aspect as reported here is the method for the manufacture of the pharmaceutical preparation comprised as antibody reported here.In yet another aspect, provide preparation, such as pharmaceutical preparation, described preparation contain prepare together with pharmaceutical carrier as antibody reported here.

Preparation of the present invention can be used by multiple method known in the art.Those of skill in the art will understand, and the approach used and/or pattern will depend on desired result and become.In order to use as compound reported here by some route of administration, may need compound with preventing the material dressing of its inactivation or using altogether with this material.Such as, this compound can be applied to experimenter in suitable carrier such as liposome or thinner.Pharmaceutically useful thinner comprises salt solution and aqueous buffer solution.Pharmaceutical carrier comprises aseptic aqueous solution or dispersion and the sterilized powder for the agent of in situ preparation sterile injectable solution or dispersion agent.Known in the art for this type of medium of pharmaceutically active substances and the purposes of material.

Many possible delivery modality can be used, include but not limited to that intraocular applies or local applies.In one embodiment, applying is that intraocular applies and comprises, but be not limited to, subconjunctival injection, intracanieral inject, are injected into anterior chamber, animalmodel by termporai edge, injection in cornea, subretinal injection, aqueous humor injection, subtenon injection or extended delivery device, intravitreal injection (such as, vitreum front portion, middle part or rear portion injection).In one embodiment, applying is local and includes, but are not limited to drop on cornea.

In one embodiment, by applying in vitreum, such as, by intravitreal injection, use as bi-specific antibody reported here or pharmaceutical preparation.This can carry out according to standard method known in the art (see, such as, the people such as Ritter, J.Clin.Invest.116 (2006) 3266-3276; The people such as Russelakis-Carneiro, the people such as Neuropathol.Appl.Neurobiol.25 (1999) 196-206 and Wray, Arch.Neurol.33 (1976) 183-185).

In some embodiments, the bi-specific antibody that can exist in pharmaceutical preparation as herein described containing one or more dosage as therapeutic agent box reported here, for this pharmaceutical preparation of intravitreal injection appropriate device and applicable object and the explanation for implementing the scheme of injecting are described in detail in detail.In these embodiments, generally preparation is applied to the experimenter needing treatment by intravitreal injection.This can carry out according to standard method known in the art (see, such as, the people such as Ritter, J.Clin.Invest.116 (2006) 3266-3276; The people such as Russelakis-Carneiro, the people such as Neuropathol.Appl.Neurobiol.25 (1999) 196-206 and Wray, Arch.Neurol.33 (1976) 183-185).

These preparations also can containing adjuvant as sanitas, wetting agent, emulsifying agent and dispersion agent.Can, by sterilization method above with by including multiple antiseptic-germicide and anti-mycotic agent in such as, guarantee to prevent there is microorganism to this manthanoate of hydroxyl, butylene-chlorohydrin, phenol, Sorbic Acid etc.Also may want isotonic agent as sugar, sodium-chlor etc. are included in preparation.In addition, the prolongation of injectable drug form can be caused to absorb by including the material such as aluminum monostearate and the gelatin that postpone to absorb in.

No matter select which kind of route of administration, all by compound (it can use by suitable hydrated form) such as reported here and/or as pharmaceutical preparation reported here, be mixed with pharmaceutically useful formulation by ordinary method well known by persons skilled in the art.

Can change the actual dose level of activeconstituents in the pharmaceutical preparation as reported herein, to obtain the following amounts of activeconstituents, described amount effectively can realize the therapeutic response wanted with regard to concrete patient, preparation and method of application, and simultaneously to patient's nontoxicity.The dosage level selected will depend on multiple pharmacokinetics factor, comprise the factor that the medical fields such as the activity of particular formulations used, route of administration, time of application, the discharge rate of specific compound used, the extended period for the treatment of, the other drug, compound and/or the material that use with agents used, age of patient for the treatment of, sex, weight, the patient's condition, general health and the past medical history are known.

Preparation must be aseptic and so flow to degree, thus by injector delivery preparation.In addition to water, carrier is preferably isotonic buffer salt brine solution.

Can such as by use dressing as Yelkin TTS, in dispersion situation by maintaining the granularity that requires and by using tensio-active agent, maintaining suitable mobility.In many cases, preferably comprise isotonic agent in the formulation, such as sugar, polyvalent alcohol are as N.F,USP MANNITOL or sorbyl alcohol and sodium-chlor.

Preparation can comprise the ophthalmic depot formulation (ophthalmicdepotformulation) comprising active substance for sub-conjunctival administration.Ophthalmic depot formulation comprises the micropartical of substantially pure active substance (such as, as bi-specific antibody reported here).The micropartical comprised as bi-specific antibody reported here can embed in biocompatibility pharmaceutical acceptable polymer or lipid encapsulated dose.The time period that depot formulation can be adapted to go through prolongation discharges all or substantially whole active substances.If existed, polymkeric substance or lipidic matrix can be adapted to degrade fully, all or substantially all to transport out from site of administration after active substance in release.Depot formulation can be liquid formulation, comprises the active substance of pharmaceutical acceptable polymer and dissolving or dispersion.Once injection, then polymkeric substance forms bank in injection site, such as, formed by gel or precipitation process.

Another aspect as reported here be used for the treatment of Ocular Vessels disease as bi-specific antibody reported here.

Another aspect as reported here be used for the treatment of Ocular Vessels disease as pharmaceutical preparation reported here.

If reported here aspect is the purposes that antibody manufacture as reported here is used for the treatment of the medicine of Ocular Vessels disease.

Another aspect as reported here is the method for the treatment of the patient suffering from Ocular Vessels disease in the following manner: use as antibody reported here to needing the patient of this treatment.

Here clearly state, comprise as " comprised " as the term is employed herein term " by ... composition ".Therefore, the whole aspect " comprised " containing term and embodiment similarly use term " by ... composition " open.

Modify

In yet another aspect, the arbitrary feature as described in hereafter 1-6 part can be merged separately or in a joint manner according to the antibody of any one above embodiment:

1. affinity of antibody

In certain embodiments, antibody provided herein has≤100nM ,≤10nM (such as 10 ^-7m or less, such as, from 10 ^-7m to 10 ^-13m, such as, from 10 ^-8m to 10 ^-13m) dissociation constant (Kd).

According to another embodiment, use surface plasmon resonance assays measures Kd.Such as, use or the assay method of (GEHealthcareInc., Piscataway, NJ) adopts immobilized antigen CM5 chip to carry out with about 10 responses unit (RU) at 25 DEG C.In one embodiment, according to supplier's specification sheets, carboxymethyl dextran resin biologic sensor chip (CM5 is activated with hydrochloric acid N-ethyl-N'-(3-dimethylamino-propyl)-carbodiimide (EDC) and N-hydroxy-succinamide (NHS), GEHealthcare, Inc.).Antigen 10mM sodium acetate, pH4.8 is diluted to 5 μ g/mL (~ 0.2 μM), replys unit (RU) subsequently with the flow velocity loading of 5 μ L/ minutes with about 10 that realize coupling protein.After injections of antigens, inject 1M thanomin with closed unreacted radical.For kinetic measurement, twice serial dilution thing (0.78nM to 500nM) of Fab to be injected in the flow velocity of about 25 μ L/ minutes at 25 DEG C there is 0.05% polysorbate20 (Tween-20 ^tM) tensio-active agent PBS in (PBST).Use simple Lang Gemiaoer combination model one to one ( evaluation software 3.2 editions) associated and the sensing figure that dissociates (sensorgram) by matching simultaneously, calculate association rate (k _on) and dissociation rate (k _off).Equilibrium dissociation constant (Kd) is calculated as ratio k _off/ k _on(see such as Chen, Y.etal., J.Mol.Biol.293 (1999) 865-881).If determine Faxian by above surface plasmon resonance measurement to show that association rate is more than 10 ⁶m-1 _s-1, then association rate can use quenching of fluorescence technical measurement, wherein said quenching of fluorescence technology at such as spectrograph (as being equipped with the spectrophotometer (AvivInstruments) of fluid stopping method (stop-flow) or there is the 8000-series SLM-AMINCO of stirring-type cuvette ^tMspectrophotometer (ThermoSpectronic)) under the antigen of measured increase concentration exists, (=295nm is excited in the fluorescent emission intensity of 25 DEG C of anti-antigen-antibodies of 20nM (Fab form) measured in PBS, pH7.2; Transmitting=340nm, 16nm band is logical) increase or reduce.

2. chimeric and humanized antibody

In certain embodiments, antibody provided herein is chimeric antibody.Some chimeric antibody such as at US4,816,567; And Morrison, S.L., wait people, Proc.Natl.Acad.Sci.USA81 (1984) 6851-6855) middle description.In one example in which, chimeric antibody comprises non-human variable region (such as, being derived from mouse, rat, hamster, rabbit or the non-human primates variable region as monkey) and human constant regions.In another example, chimeric antibody is " class conversion " antibody, wherein class or subclass relatively parental antibody change.Chimeric antibody comprises its antigen in conjunction with its fragment.

In certain embodiments, chimeric antibody is humanized antibody.Usually, by non-human antibody's humanization to reduce the immunogenicity to people, retain specificity and the avidity of parent non-human antibody simultaneously.Usually, humanized antibody comprises wherein HVR such as CDR (or its part) and is derived from non-human antibody and FR (or its part) is derived from one or more variable domains of human antibody sequence.Humanized antibody optionally also will comprise human constant region at least partially.In some embodiments, be the corresponding residue from non-human antibody's (such as, the therefrom antibody of derivative HVR residue) by some the FR residue substitutions in humanized antibody, such as, to recover or to improve antibodies specific or avidity.

Humanized antibody and their method of manufacture are such as at Almagro, JAlmagro, J.C. and Fransson, J., summarize in Front.Biosci.13 (2008) 1619-1633, and such as at Riechmann, I. people is waited, Nature332 (1988) 323-329; The people such as Queen, C., Proc.Natl.Acad.Sci.USA86 (1989) 10029-10033; US5,821,337, US7,527,791, US6,982,321 and US7,087,409; The people such as Kashmiri, S.V., Methods36 (2005) 25-34 (describing specificity determining area (SDR) to transplant); Padlan, E.A., Mol.Immunol.28 (1991) 489-498 (describing " surface is reinvented (resurfacing) "); The people such as Dall'Acqua, W.F., Methods36 (2005) 43-60 (describing " FR reorganization "); The people such as Osbourn, J., Methods36 (2005) 61-68; And the people such as Klimka, A., further describe in Br.J.Cancer83 (2000) 252-260 (describing " guided selection " scheme for FR reorganization).

May be used for humanized people framework region to include but not limited to: the framework region (such as, seeing Sims, the people such as M.J., J.Immunol.151 (1993) 2296-2308) using " best fit " method choice; Carter (such as, is seen, the people such as P., Proc.Natl.Acad.Sci.USA89 (1992) 4285-4289 in the framework region derived from the variable region of light chain of people's antibody of specific subgroup or the consensus sequence of variable region of heavy chain; And the people such as Presta, L.G., J.Immunol.151 (1993) 2623-2632); People's maturation (somatic mutation) framework region or human germline framework (such as, seeing Almagro, J.C. and Fransson, J., Front.Biosci.13 (2008) 1619-1633); With the framework region (such as, see Baca, the people such as M., the people such as J.Biol.Chem.272 (1997) 10678-10684 and Rosok, M.J., J.Biol.Chem.271 (1996) 22611-22618) derivative from screening FR library.

3. people's antibody

In certain embodiments, antibody provided herein is people's antibody.Multiple technologies known in the art can be used to produce people's antibody.At vanDijk, M.A. and vandeWinkel on people's antibody population, describe in J.G., Curr.Opin.Pharmacol.5 (2001) 368-374 and Lonberg, N., Curr.Opin.Immunol.20 (2008) 450-459.

Can prepare people's antibody by using immunogen to transgenic animal, wherein said transgenic animal have been modified to and have responded to antigen and attack and produce complete human antibody or have the complete antibody of people variable region.This kind of animal is generally containing replacing endogenous immunoglobulin locus or existing outward or random integration enters the chromosomal all or part of human immunoglobulin gene's seat of animal at karyomit(e).In this kind of transgenic mice, the usual inactivation of endogenous immunoglobulin locus.About the summary of method obtaining people's antibody from transgenic animal, see Lonberg, N., Nat.Biotech.23 (2005) 1117-1125.Also see, such as, describe XENOMOUSE ^tMthe US6 of technology, 075,181 and US6,150,584; Describe the US5 of technology, 770,429; K-M is described the US7 of technology, 041,870, and Veloci is described the US2007/0061900 of technology).The people variable region of the complete antibody produced from this kind of animal can be modified further, such as, by combining from different human constant regions.

Also people's antibody can be produced by the method based on hybridoma.Describe and (such as, seen Kozbor, D., J.Immunol.133 (1984) 3001-3005 for generation of the human myeloma of human monoclonal antibodies and mouse-human heteromyeloma's clone; The people such as Brodeur, B.R., MonoclonalAntibodyProductionTechniquesandApplications, MarcelDekker, Inc., NewYork (1987), 51-63 page; And the people such as Boerner, P., J.Immunol.147 (1991) 86-95).People such as Li, J., in Proc.Natl.Acad.Sci.USA103 (2006) 3557-3562, also illustrate the people's antibody produced by people B-cell hybridoma technique.Extra method comprises such as at US7,189,826 (they describe and produce monoclonal human IgM antibody from hybridoma cell line) and middle those methods described of Ni, J., XiandaiMianyixue26 (2006) 265-268 (it describes people-people's hybridoma).At Vollmers, and Brandlein H.P., S., Histology and Histopathology20 (2005) 927-937 and Vollmers, and Brandlein H.P., S., people's hybridoma technology (Trioma technology) is also illustrated in MethodsandFindingsinExperimentalandClinicalPharmacology2 7 (2005) 185-191.

Also variable domain sequence generation people antibody can be cloned by being separated the Fv selected from the phage display library that people derives.This kind of variable domain sequence can combine with required people's constant domain subsequently.The technology being used for selecting people's antibody from antibody library is hereafter described.

4. the antibody that library is derivative

By having the antibody of one or more activity required to combinatorial library screening, can be separated as antibody reported here.Such as, known in the art have for generation of phage display library and to this kind of library screening needed in conjunction with the multiple method of the antibody of feature.These class methods such as people such as Hoogenboom, H.R., in MethodsinMolecularBiology178 (2001) 1-37 summary and such as people such as McCafferty, J., Nature348 (1990) 552-554; The people such as Clackson, T., Nature352 (1991) 624-628; The people such as Marks, J.D., J.Mol.Biol.222 (1992) 581-597; Marks, J.D. and Bradbury, A., MethodsinMolecularBiology248 (2003) 161-175; The people such as Sidhu, S.S., J.Mol.Biol.338 (2004) 299-310; The people such as Lee, C.V., J.Mol.Biol.340 (2004) 1073-1093; The people such as Fellouse, F.A., Proc.Natl.Acad.Sci.USA101 (2004) 12467-12472 and Lee, C.V., further describe in J.Immunol.Methods284 (2004) 119-132.

In some phage display, VH gene and VL gene pool are cloned by polymerase chain reaction (PCR) respectively and are recombinated at random in phage library, wherein subsequently can to the phage of described phage library screening conjugated antigen, as Winter, G., Deng people, Ann.Rev.Immunol., described in 12 (1994) 433-455.Antibody fragment is generally shown as scFv (scFv) fragment or is shown as Fab fragment by phage.The control oneself library of immune origin provides for immunogenic high-affinity antibody, without the need to building hybridoma.Alternatively, can (such as, from people) clone natural storehouse with when not carrying out any immunity, there is provided for the antibody of the non-self antigen of wide range of types with also for the single source of the antibody of autoantigen, as Griffiths, A.D., wait people, described in EMBOJ.12 (1993) 725-734.Finally, also can use and realize the PCR primer of external rearrangement containing stochastic sequence with the variable CDR3 district of code level by the V-constant gene segment C do not reset from stem cell clone, produce naive libraries synthetically, as Hoogenboom, and Winter H.R., G., described in J.Mol.Biol.227 (1992) 381-388.The patent disclosure describing people's antibody phage libraries such as comprises: US5,750,373 and US2005/0079574, US2005/0119455, US2005/0266000, US2007/0117126, US2007/0160598, US2007/0237764, US2007/0292936 and US2009/0002360.

Think that the antibody that is separated from people's antibody library or antibody fragment are people's antibody herein or people's antibody fragment.

5. multi-specificity antibody

In certain embodiments, antibody provided herein is multi-specificity antibody, such as bi-specific antibody.Multi-specificity antibody is the monoclonal antibody at least two different loci to binding specificity.Bi-specific antibody also can be used for making cytotoxic agent be limited to the cell of expressing one or more target antigens.Bi-specific antibody can be prepared as full length antibody or antibody fragment.

Technology for generation of multi-specificity antibody includes but not limited to that recombinant co-expression has not homospecific two heavy chain immunoglobulin-light chains to (see Milstein, and Cuello C., A.C., Nature305 (1983) 537-540, WO93/08829 and Traunecker, the people such as A., EMBOJ.10 (1991) 3655-3659) and " projection-enter-hole " through engineering approaches is (such as, see US5,731,168).Also multi-specificity antibody can be produced in the following manner: through engineering approaches electrostatic manipulation effects is for generation of antibody Fc-heterodimeric molecule (WO2009/089004); Two or more antibody crosslinked or fragment (such as, seeing US4,676,980 and the people such as Brennan, M., Science229 (1985) 81-83); Use leucine zipper to produce bi-specific antibody (such as, seeing Kostelny, the people such as S.A., J.Immunol.148 (1992) 1547-1553); " Diabody (the diabody) " technology of use is to produce bispecific antibody fragment (such as, seeing Holliger, the people such as P., Proc.Natl.Acad.Sci.USA90 (1993) 6444-6448); With use scFv (sFv) dimer (such as, seeing Gruber, the people such as M., J.Immunol.152 (1994) 5368-5374); And prepare three-specific antibody, and as people such as such as Tutt, A., J.Immunol.147 (1991) 60-69) described in.

Also comprise the engineered antibody with three or more functional antigen binding sites herein, comprise " eight antibody (Octopusantibody) " (such as, see, US2006/0025576).

Antibody herein or fragment also comprise " dual-use function Fab " or " DAF ", and it comprises and the first antigen and the another kind not antigen binding site (for example, see, US2008/0069820) that is combined of synantigen.

Antibody herein or fragment are also included within the multi-specificity antibody described in WO2009/080251, WO2009/080252, WO2009/080253, WO2009/080254, WO2010/112193, WO2010/115589, WO2010/136172, WO2010/145792 and WO2010/145793.

6. antibody variants

In certain embodiments, the amino acid sequence variation of antibody provided herein is contemplated.Such as, binding affinity and/or the other biological characteristic of improving antibody may be wanted.Can by introducing the suitable amino acid sequence variation modified or pass through peptide symthesis Dispersal risk to the nucleotide sequence of encoding antibody.This type of modification comprises such as, from the inner deleting residues of the aminoacid sequence of antibody and/or inserted by residue described aminoacid sequence and/or the residue of replacing in described aminoacid sequence.Can produce disappearance, insert and displacement arbitrary combination to obtain final construct, as long as described final construct has the feature wanted, such as antigen keying action.

A) replace variant, insert variant and deletion mutants

In certain embodiments, the antibody variants with one or more amino-acid substitution is provided.Object site for replacing mutagenesis comprises HVR and FR.Under " preferably replacing " title, preservative replacement is shown in following table.Show under " exemplary permutation " title in following table and more obviously change as described further below with reference to amino acid side chain classification.Amino-acid substitution can be introduced in object antibody and to the activity needed for product screening, such as, the antigen keying action retaining/improve or the immunogenicity of reduction, or ADCC or CDC improved.

table

Original Residue	Exemplary permutation	Preferred displacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp、Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu, Val; Met; Ala; Phe; Nor-leucine	Leu
			Leu(L)	Nor-leucine; Ile; Val; Met; Ala; Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; Leu; Met; Phe; Ala; Nor-leucine	Leu

Amino acid can divide into groups according to common side chain properties:

(1) hydrophobicity: nor-leucine, Met, Ala, Val, Leu; Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn; Gln;

(3) acid: Asp, Glu;

(4) alkalescence: His, Lys, Arg;

(5) residue of chain direction is affected: Gly, Pro;

(6) aromatics: Trp, Tyr, Phe.

The member one of making these classify is exchanged for another member classified by non-conservation displacement.

A displacement variant type relates to one or more some hypervariable region residues of displacement parental antibody (such as, humanization or people's antibody).Usually, gained variant through selecting to be used for further research relative to parental antibody in some biological characteristics (such as, the avidity increased, the immunogenicity of reduction) there is modification (such as, improving) and/or will some biological characteristics substantially retained of parental antibody be had.Exemplary permutation variant is affinity maturation antibody, and described antibody can such as, use based on phage display affinity maturation technology as those described herein technology produce expediently.In brief, show on phage by one or more HVR residue mutations and by variant antibodies and screen particular organisms activity (such as binding affinity).

Can make a change in HVR (such as, displacement), such as, to improve affinity of antibody.This kind of change can at HVR " focus " (namely, during somatocyte ripening process with high frequency experience sudden change codon coded by residue (see, such as, Chowdhury, P.S., MethodsMol.Biol.207 (2008) 179-196) and/or contact antigen residue in make, simultaneously to produced variant VH or VL test binding affinity.Such as people such as Hoogenboom, H.R., drawn from MethodsinMolecularBiology178 (2002) 1-37) in describe by building secondary library and therefrom reselecting and realize affinity maturation.In some embodiments of affinity maturation, by any one of multiple method (such as, the reorganization of fallibility PCR, chain or oligonucleotide directed mutagenesis), be used in ripe variable gene selected by diversity is introduced.Produce secondary library subsequently.Screen this library has required avidity any antibody variants with qualification subsequently.The another kind of scheme introduced multifarious method and relate to HVR guidance, wherein by several HVR residue (such as, a 4-6 residue) random packet.The HVR residue participating in antigen and combine can be identified especially, such as, use Alanine-scanning method mutagenesis or modeling.Frequent target CDR-H3 and CDR-L3 especially.

In certain embodiments, displacement, to insert or disappearance can occur in one or more HVR inside, if this kind of change not essence reduce the ability of antibodies bind antigen.Such as, conservative property change (preservative replacement such as, as provided) that not essence reduces binding affinity can be made herein in HVR.This kind of change, such as, can in the HVR " outside of antigen contact residue.In some embodiment of variant VH provided above and VL sequence, each HVR will not change or containing no more than one, two or three amino-acid substitutions.

A kind of for the identification of being targeted so that the process useful in the antibody residue of mutagenesis or region is called " Alanine-scanning method mutagenesis ", as Cunningham, B.C. and Wells, J.A., Science, described in 244 (1989) 1081-1085.In this approach, identify a residue or target residue group (such as, charged residue is as arg, asp, his, lys and glu) and replace whether influenced to determine the interaction of this antibody and antigen with neutral or electronegative amino acid (such as, L-Ala or polyalanine).Other displacements can be introduced at the amino acid position place for initial permutation Presentation Function sensitivity.Alternatively or extraly, the crystalline structure of antigen-antibody complex can be used to identify the point of contact between antibody and antigen.This kind of contact residues and contiguous residue can be practiced shooting or elimination as replacement candidate thing.Variant can be screened to determine that whether they are containing required characteristic.

Aminoacid sequence inserts and comprises length from 1 residue to containing into the aminoterminal that changes between the polypeptide of hundred or more residues and/or carboxyl terminal merges, and inserts in the sequence of single or multiple amino-acid residue.The example that end inserts comprises the antibody with N-terminal methionyl residue.Other property inserted variants of antibody molecule comprise the N-terminal of antibody or C-terminal and enzyme (such as the enzyme of ADEPT) or increase the peptide fusion of this antibody serum half life.

B) glycosylation variants

In certain embodiments, change antibody provided herein, to increase or to reduce antibody, glycosylated degree occurs.By changing aminoacid sequence thus creating or remove one or more glycosylation site, antagonist can be realized expediently and adds or delete glycosylation site.

When antibody comprises Fc district, the sugar be attached thereto can be changed.The natural antibody that mammalian cell produces generally comprises two feeler oligose of branch, and described oligose is connected to the Asn297 of the CH2 structural domain in Fc district usually by N connection.See, such as, Wright, A. and Morrison, S.L., TIBTECH15 (1997) 26-32.Oligose can comprise various sugar, such as, and seminose, 2-Acetamido-2-deoxy-D-glucose (GlcNAc), semi-lactosi and sialic acid, and the Fucose be connected with GlcNAc in " stem " of two feeler oligosaccharide structures.In some embodiments, the oligose can modified in the antibody as reported herein improves the antibody variants of some characteristic with generation.

In one embodiment, provide the antibody variants with sugared structure, described sugared structure lacks the Fucose be connected with Fc district (directly or indirectly).Such as, in this antibody-like, the amount of Fucose can be 1% to 80%, 1% to 65%, 5% to 65% or 20% to 40%.Determine the amount of Fucose in the following manner: relative to the summation such as passing through the whole sugared structure (such as complex construction, hybrid structure and high mannose structures) be connected with Asn297 measured by MALDI-TOF mass spectroscopy, calculate the mean vol of sugar chain inner Asn297 place Fucose, such as, as described in WO2008/077546.Asn297 refers to the asparagine residue (the EU numbering of Fc district residue) being positioned at position, Fc district the about the 297th; But near the upstream that Asn297 also can be positioned at the 297th position or downstream ± 3 amino acid, that is, between the 294th and 300 positions, reason is the minor sequence variation in antibody.This kind of fucosylation variant can have the ADCC function of improvement.See, such as, US2003/0157108; US2004/0093621.Comprise to the example of " go fucosylated " or " lacking Fucose " publication that antibody variants is relevant: US2003/0157108; WO2000/61739; WO2001/29246; US2003/0115614; US2002/0164328; US2004/0093621; US2004/0132140; US2004/0110704; US2004/0110282; US2004/0109865; WO2003/085119; WO2003/084570; WO2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; The people such as Okazaki, A., J.Mol.Biol.336 (2004) 1239-1249; The people such as Yamane-Ohnuki, N., Biotech.Bioeng.87 (2004) 614-622.The example that can produce the clone of fucosylated antibody is included in the Lec13CHO cell of protein fucosylated aspect defect (people such as Ripka, J., Arch.Biochem.Biophys.249 (1986) 533-545; US2003/0157108; And WO2004/056312, especially at embodiment 11 place) and knock out clone, as α-1,6-fucosyl transferase gene FUT8 knocks out Chinese hamster ovary celI (such as, see people such as Yamane-Ohnuki, N., Biotech.Bioeng.87 (2004) 614-622; The people such as Kanda, Y., Biotechnol.Bioeng.94 (2006) 680-688; And WO2003/085107).

Two points of oligose can also be provided for antibody variants, such as, the two feeler oligose is wherein connected with antibody Fc district by GlcNAc to dividing.The ADCC function that this kind of antibody variants can have the fucosylated of minimizing and/or improve.The example of this kind of antibody variants is such as at WO2003/011878; US6,602,684; Describe with in US2005/0123546.The antibody variants that in oligose, at least one galactose residue is connected with Fc district is also provided.This kind of antibody variants can have the CDC function of improvement.This kind of antibody variants is such as at WO1997/30087; Describe in WO1998/58964 and WO1999/22764.

C) Fc region variants

In certain embodiments, can one or more other be amino acid modifiedly incorporated herein in the Fc district of the antibody provided, thus produce Fc region variants.This Fc region variants can comprise people Fc region sequence (such as, human IgG1, IgG2, IgG3 or IgG4Fc district), and described people Fc region sequence comprises at one or more amino acid position place amino acid modified (such as, replacing/sudden change).

In certain embodiments, this invention contemplates and have some but the antibody variants of not all effector function, this makes described antibody variants become the favourable material standed for of following application, wherein the Half-life in vivo of antibody is important, and some effector function (as CDC and ADCC) is unnecessary or harmful.Can the outer and/or in vivo cytotoxicity assay method of embodiment to confirm that CDC and/or ADCC activity reduces/exhausts.Such as, Fc acceptor (FcR) binding assay can be implemented to guarantee that antibody lacks Fc γ R keying action (therefore may lack ADCC activity), but retain FcRn binding ability.Primary cell-NK the cell of mediation ADCC only expresses Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.Ravetch, J.V. and Kinet, the FcR summarized in the table 3 on the 464th page of J.P., Annu.Rev.Immunol.9 (1991) 457-492 on hematopoietic cell expresses.The non-limitative example of the vitro assay of the ADCC activity of purpose of appraisals molecule at US5,500,362 (see, such as, the people such as Hellstrom, I., Proc.Natl.Acad.Sci.USA83 (1986) 7059-7063; And the people such as Hellstrom, I., Proc.Natl.Acad.Sci.USA82 (1985) 1499-1502); US5,821,337 (see people such as Bruggemann, M., J.Exp.Med.166 (1987) 1351-1361)) middle description.Alternatively, can use on-radiation analytical procedure (see, such as, for the ACTI of flow cytometry ^tMnon-radioactive cell toxicity assay (CellTechnology, Inc.MountainView, CA; And CytoTox non-radioactive cell toxicity assay (Promega, Madison, WI).Effector cell for this type of assay method draws together peripheral blood lymphocytes (PBMC) and NK cell (NK) cell.Alternative or extraly, can in vivo, such as, in animal model, as people such as Clynes, R., in that animal model during Proc.Natl.Acad.Sci.USA95 (1998) 652-656 is open, the ADCC of purpose of appraisals molecule is active.Also C1q binding assay can be implemented to confirm that therefore antibody also can not lack CDC activity in conjunction with C1q.See, such as, C1q and C3c in WO2006/029879 and WO2005/100402 is in conjunction with ELISA.In order to assess complement activation, can carry out CDC assay method (see, such as, the people such as Gazzano-Santoro, H., J.Immunol.Methods202 (1996) 163-171; The people such as Cragg, M.S., Blood101 (2003) 1045-1052; And Cragg, M.S. and M.J.Glennie, Blood103 (2004) 2738-2743).Also can use methods known in the art determine in FcRn keying action and body clearance rate/transformation period (see, such as, the people such as Petkova, S.B., Int.Immunol.18 (2006) 1759-1769).

Effector function reduce antibody comprise displacement one or more Fc districts residue 238,265,269,270,297,327 and 329 (US6,737,056) those.Two or more positions that this kind of Fc region variants is included in amino acid position 265,269,270,297 and 327 have the Fc district of displacement, comprise what is called " DANA " the Fc region mutation body (US7 that residue 265 and 297 is replaced as L-Ala, 332,581).

Describe have FcR keying action improve or weaken some antibody variants (see, such as, US6,737,056; WO2004/056312, and the people such as Shields, R.L., J.Biol.Chem.276 (2001) 6591-6604).

In certain embodiments, antibody variants comprises the Fc district with the one or more amino-acid substitutions (such as, in the displacement of the 298th, 333 and/or 334 positions in Fc district) (the EU numbering of residue) improving ADCC.

In some embodiments, make a change in Fc district, described change causes the C1q keying action and/or the complement dependent cytotoxicity (CDC) that change (namely improve or weaken), such as, as US6,194,551, WO99/51642 and Idusogie, E.E. people is waited, described in J.Immunol.164 (2000) 4178-4184.

The antibody that the transformation period increases and neonatal Fc receptor (FcRn) keying action is improved is described in US2005/0014934, described neonatal Fc receptor is responsible for transfer source of parents IgG to fetus (Guyer, R.L. people is waited, J.Immunol.117 (1976) 587-593 and Kim, J.K. people is waited, J.Immunol.24 (1994) 2429-2434).These antibody comprise the Fc district wherein with one or more displacement, and wherein said displacement improves the combination of Fc district and FcRn.This kind of Fc region variants is included in one or more Fc districts residue: 238,256,265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424 or 434 places have displacement (such as, the displacement of Fc district residue 434) those (US7,371,826).

About other examples of Fc region variants, also see Duncan, A.R. and Winter, G., Nature322 (1988) 738-740; US5,648,260; US5,624,821 and WO94/29351.

D) halfcystine engineered antibody variant

In certain embodiments, may want the antibody producing halfcystine through engineering approaches, such as, " sulfo-MAb ", wherein one or more residue cysteine residues of antibody are replaced.In particular embodiments, the residue of displacement appears at the Accessibility site place of antibody.By replacing these residues with halfcystine, thus reactive sulfydryl be placed in the Accessibility site place of antibody and can be used for making antibody conjugate to other parts, if drug moiety or linker-drug part are to produce immunoconjugates, as further described herein.In certain embodiments, any one or more of following residue can be replaced with halfcystine: the V205 (Kabat numbering) of light chain and the A118 (EU numbering) of heavy chain; And the S400 in heavy chain Fc district (EU numbering).Can as US7,521, produce the antibody of halfcystine through engineering approaches described in 541 like that.

E) antibody derivatives

In certain embodiments, antibody provided herein can be modified further with containing known in the art and obtainable extra non-protein portion easily.The part being suitable for derivative antibody includes but not limited to water-soluble polymers.The non-limitative example of water-soluble polymers includes but not limited to multipolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-DOX, poly-1,3, the 6-tri-of polyoxyethylene glycol (PEG), ethylene glycol/propylene glycol alkane, ethylidene/maleic anhydride copolymers, polyamino acid (homopolymer or random copolymers) and dextran or poly-(n-vinyl pyrrolidone) polyoxyethylene glycol, propropylene glycol homopolymers, poly(propylene oxide)/ethylene oxide copolymer, oxyethylated polyols (such as, glycerol), polyvinyl alcohol and their mixture.Methoxy PEG-propionaldehyde can have the advantage of manufacture view, and reason is its stability in water.This polymkeric substance can have any molecular weight, and can be branch or not branch.The number of the polymkeric substance be connected with antibody can change, and if connect more than one polymkeric substance, they can be identical or different molecules.Usually, can determine based on following consideration item for the number of the polymkeric substance of derivatize and/or type, include but not limited to antibody particular characteristics to be improved or function, antibody derivatives whether by the therapy that is used in restriction situation etc.

In another embodiment, antibody and can by being exposed to the conjugate of non-protein portion of radiation and selectivity heating is provided.In one embodiment, non-protein portion is carbon nanotube (people such as Kam, N.W., Proc.Natl.Acad.Sci.USA102 (2005) 11600-11605).Radiation can have any wavelength, and includes, but are not limited to such wavelength, and described wavelength does not injure ordinary cells, but makes nonprotein portion be heated to kill and wound the temperature of the cell being close in antibody-non-protein portion.

F) different dimerization

There is several modification CH3 to strengthen the scheme of different dimerization, these schemes such as fully describe in WO96/27011, WO98/050431, EP1870459, WO2007/110205, WO2007/147901, WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012058768, WO2013157954, WO2013096291.Generally, in whole this kind of scheme, one CH3 structural domain and the 2nd CH3 structural domain through engineering approaches all in complementary fashion, thus each CH3 structural domain (or comprising its heavy chain) can not more muchly with self Homodimeric, but to be forced to and another CH3 structural domain different dimerization (thus the first and second CH3 structural domain different dimerizations and do not form homodimer between two CH3 structural domains or between two the 2nd CH3 structural domains) of complementary through engineering approaches.Conceive these different schemes for improving heavy chain different dimerization as modifying the different replacement schemes (in a brachium conjunctivum VH and VL exchange/replace and the charge residue introducing oppositely charged in CH1/CL interface is replaced) combine in multi-specificity antibody of the present invention from Chong Lian – light chain, the Bence-Jones type by product that this minimizing light chain mistake is matched.

In a preferred embodiment of the invention (in this case, multi-specificity antibody comprises CH3 structural domain in heavy chain), can be changed the CH3 structural domain of described multi-specificity antibody of the present invention by " projection-enter-hole " technology, described technology describes in detail with such as WO96/027011; The people such as Ridgway, J.B., ProteinEng.9 (1996) 617-621; And the people such as Merchant, A.M., Nat.Biotechnol.16 (1998) 677-681; Several example in detail in WO98/050431.In this approach, the interaction face of two CH3 structural domains is changed to increase the different dimerization of two heavy chains containing these two CH3 structural domains.The one of (two heavy chain) two CH3 structural domains can be " projection ", and another one is " hole ".Heterodimer (people such as Merchant, A.M, NatureBiotech.16 (1998) 677-681 are stablized in the introducing of disulfide linkage further; The people such as Atwell, S., J.Mol.Biol.270 (1997) 26-35) and increase productive rate.

Therefore in one embodiment of the invention, described multi-specificity antibody (comprise in every bar heavy chain CH3 structural domain and) is further characterized in that

Join in the interface that each leisure of 2nd CH3 structural domain of the second heavy chain of the one CH3 structural domain of the first heavy chain of the antibody under a) and the antibody under b) comprises initial interface between antibody CH3 structural domain.

Wherein change described interface to promote the formation of multi-specificity antibody, wherein said change is characterised in that

I) the CH3 structural domain of a heavy chain is so changed,

Thus at multi-specificity antibody the CH3 structural domain of an inner heavy chain of joining with the initial interface of the CH3 structural domain of another heavy chain initial interface in,

Amino-acid residue is replaced with the amino-acid residue with more bulky side chain volume, thus produce protuberance in the interface internal of the CH3 structural domain of a heavy chain, described protuberance can be placed in the chamber of the interface internal of the CH3 structural domain of another heavy chain.

And

Ii) the CH3 structural domain of another heavy chain is so changed,

Thus inner at multi-specificity antibody, in the initial interface of the 2nd CH3 structural domain of joining with the initial interface of a CH3 structural domain,

Amino-acid residue is replaced with the amino-acid residue with less side-chain bulk, thus produce chamber in the interface internal of the 2nd CH3 structural domain, the protuberance can placing the interface internal of a CH3 structural domain in inside, described chamber can be arranged in.

Preferably, the amino-acid residue described in more bulky side chain volume is selected from arginine (R), phenylalanine (F), tyrosine (Y), tryptophane (W).

Preferably, the amino-acid residue described in less side-chain bulk is selected from L-Ala (A), Serine (S), Threonine (T), α-amino-isovaleric acid (V).

In one aspect of the invention, change two CH3 structural domains further in the following manner: so introduce halfcystine (C) as the amino acid in the corresponding position of each CH3 structural domain, thus the disulfide linkage between two CH3 structural domains can be formed.

In one embodiment, described multi-specificity antibody comprises amino acid T366W sudden change and in the 2nd CH3 structural domain of " pore chain ", comprises amino acid T366S, L368A, Y407V sudden change in a CH3 structural domain of " projection chain ".Also the extra interchain disulfide bond (Merchant between CH3 structural domain can be used, the people such as A.M, NatureBiotech16 (1998) 677-681), such as, by introducing amino acid Y349C sudden change to the CH3 structural domain of " pore chain " and introducing amino acid E356C sudden change to the CH3 structural domain of " raised chain " or amino acid S354C suddenlys change.

In a preferred embodiment, described multi-specificity antibody (it comprises CH3 structural domain in every bar heavy chain) comprises amino acid S354C, T366W sudden change and in another structural domain of two CH3 structural domains, comprises amino acid Y349C, T366S, L368A, Y407V sudden change (the additional amino acid S354C sudden change in a CH3 territory becomes interchain disulfide bond with the additional amino acid Y349C mutant form in another CH3 territory) (according to Kabat numbering) in one of two CH3 structural domains.

Conceiving for modifying CH3 using the other technologies strengthening different dimerization as of the present invention alternative and they are such as at WO96/27011, WO98/050431, describing in EP1870459, WO2007/110205, WO2007/147901, WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954, WO2013/096291.

In one embodiment, the different dimerization scheme described in EP1870459A1 can alternatively be used.This method introduces the charge residue displacement/sudden change of oppositely charged based on specific amino acids position in the CH3/CH3 domain interfaces between two heavy chains.A preferred embodiment of described multi-specificity antibody is in amino acid R409D, K370E sudden change of (multi-specificity antibody) the one in CH3 structural domain and amino acid D399K, E357K sudden change (according to Kabat numbering) in the 2nd CH3 structural domain at multi-specificity antibody.

In another embodiment, described multi-specificity antibody comprises amino acid T366W sudden change and in the CH3 structural domain of " pore chain ", comprises amino acid T366S, L368A, Y407V sudden change and in the CH3 structural domain of " projection chain ", comprise amino acid R409D, K370E sudden change extraly and in the CH3 structural domain of " pore chain ", comprise amino acid D399K, E357K sudden change in the CH3 structural domain of " projection chain ".

In another embodiment, described multi-specificity antibody comprises amino acid S354C in one of two CH3 structural domains, T366W suddenlys change and comprise amino acid Y349C in another structural domain of two CH3 structural domains, T366S, L368A, Y407V suddenlys change, or described multi-specificity antibody comprises amino acid Y349C in one of two CH3 structural domains, T366W suddenlys change and comprise amino acid S354C in another structural domain of two CH3 structural domains, T366S, L368A, Y407V suddenlys change and in the CH3 structural domain of " projection chain ", comprises amino acid R409D extraly, K370E suddenlys change and comprise amino acid D399K in the CH3 structural domain of " pore chain ", E357K suddenlys change.

In one embodiment, the different dimerization scheme described in WO2013/157953 can alternatively be used.In one embodiment, a CH3 structural domain comprises amino acid T366K and to suddenly change and the 2nd CH3 Domain Polypeptide comprises amino acid L351D suddenlys change.In still another embodiment, a CH3 structural domain comprises other amino acid L351K and suddenlys change.In still another embodiment, the 2nd CH3 structural domain comprises other amino acid mutations being selected from Y349E, Y349D and L368E (preferably L368E).

In one embodiment, the different dimerization scheme described in WO2012/058768 can alternatively be used.In one embodiment, a CH3 structural domain comprise amino acid L351Y, Y407A sudden change and the 2nd CH3 structural domain comprise amino acid T366A, K409F sudden change.In still another embodiment, 2nd CH3 structural domain comprises other amino acid mutations in T411, D399, S400, F405, N390 or K392 position, described amino acid mutation is such as selected from a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R or S400K, F405I, F405M, F405T, F405S, F405V or F405W, N390R, N390K or N390D, K392V, K392M, K392R, K392L, K392F or K392E.In still another embodiment, a CH3 structural domain comprise amino acid L351Y, Y407A sudden change and the 2nd CH3 structural domain comprise amino acid T366V, K409F sudden change.In still another embodiment, a CH3 structural domain comprise amino acid Y407A suddenly change and the 2nd CH3 structural domain comprise amino acid T366A, K409F sudden change.In still another embodiment, the 2nd CH3 structural domain comprises other amino acid mutations K392E, T411E, D399R and S400R.

In one embodiment, alternatively can use the different dimerization scheme described in WO2011/143545, such as, have amino acid modified in the position being selected from 368 and 409.

In one embodiment, alternatively can use the different dimerization scheme described in WO2011/090762, described scheme also uses projection mentioned above-enter-hole technology.In one embodiment, a CH3 structural domain comprises amino acid T366W and to suddenly change and the 2nd CH3 structural domain comprises amino acid Y407A suddenlys change.In one embodiment, a CH3 structural domain comprises amino acid T366Y and to suddenly change and the 2nd CH3 structural domain comprises amino acid Y407T suddenlys change.

In one embodiment, multi-specificity antibody belongs to IgG2 isotype and alternatively can use the different dimerization scheme described in WO2010/129304.

In one embodiment, the different dimerization scheme described in WO2009/089004 can alternatively be used.In one embodiment, one CH3 structural domain comprises electronegative amino acid (such as L-glutamic acid (E) or aspartic acid (D), preferably K392D or N392D) to the amino-acid substitution of K392 or N392 and the 2nd CH3 structural domain comprises positively charged amino acid (such as Methionin (K) or arginine (R), preferably D399K, E356K, D356K or E357K and more preferably D399K and the E356K) amino-acid substitution to D399, E356, D356 or E357.In still another embodiment, a CH3 structural domain also comprises electronegative amino acid (such as L-glutamic acid (E) or aspartic acid (D), preferably K409D or the R409D) amino-acid substitution to K409 or R409.In still another embodiment, a CH3 structural domain comprises electronegative amino acid (such as L-glutamic acid (E), or aspartic acid (the D)) amino-acid substitution to K439 and/or K370 further or additionally.

In one embodiment, the different dimerization scheme described in WO2007/147901 can alternatively be used.In one embodiment, a CH3 structural domain comprise amino acid K253E, D282K and K322D sudden change and the 2nd CH3 structural domain comprise amino acid D239K, E240K and K292D sudden change.

In one embodiment, the different dimerization scheme described in WO2007/110205 can alternatively be used.

Recombination method and preparation

Recombination method and preparation can be used to produce antibody, such as, as US4,816, described in 567.In one embodiment, the isolating nucleic acid of antibody as described herein of encoding is provided.This nucleic acid can encoded packets containing antibody VL aminoacid sequence and/or comprise the aminoacid sequence (such as, the light chain of antibody and/or heavy chain) of antibody VH.In still another embodiment, one or more carriers comprising this nucleic acid (such as, expression vector) are provided.In still another embodiment, providing package is containing the host cell of this nucleic acid.In such embodiment, host cell comprises (such as, transform with it): (1) comprises the carrier of nucleic acid, described nucleic acid encoding comprises the aminoacid sequence of the VL of antibody and comprises the aminoacid sequence of VH of antibody, or (2) comprise encoded packets containing the first carrier of the nucleic acid of the aminoacid sequence of the VL of antibody with comprise the Second support of encoded packets containing the nucleic acid of the aminoacid sequence of the VH of antibody.In one embodiment, host cell is eucaryon, such as Chinese hamster ovary (CHO) cell or lymphoidocyte (such as, Y0, NS0, Sp20 cell).In one embodiment, a kind of method produced as antibody reported here is provided, wherein said method cultivates the host cell of the nucleic acid comprising encoding antibody as provided under being included in the condition being suitable for expressing antibody, and optionally reclaims this antibody from host cell (or host cell substratum).

Producing variant Fc district to recombinate, the nucleic acid (such as, as described above) in coding this variant Fc district being separated and inserting in one or more carriers for clone and/or expression further in host cell.Ordinary method (such as, by using the oligonucleotide probe that can be combined with the gene specific of encode variant Fc district's polypeptide or heavy chain of antibody and light chain) can being used, being separated this nucleic acid easily and being checked order.

The host cell being suitable for the carrier of cloning or expressing encoding antibody comprises protokaryon as herein described or eukaryotic cell.Such as, especially when not needing glycosylation and Fc effector function, antibody can be produced in bacterium.For antibody fragment and polypeptide, the expression in bacterium is shown in such as US5,648,237, US5,789,199 and US5,840,523 (also see Charlton, K.A., In:MethodsinMolecularBiology, 248 volumes, Lo, B.K.C. (editor), HumanaPress, Totowa, NJ (2003), 245-254 page, it describes the expression of antibody fragment in intestinal bacteria).After expression, separation antibody can be stuck with paste from the bacterial cell soluble fraction, and can be further purified.

Except prokaryotic organism, the eukaryotic microorganisms of such as filamentous fungus or yeast is also suitable clone or the expressive host of the carrier of encoding antibody, comprise glycosylation pathway differ " humanization ", cause producing the fungi and yeasts strain of the antibody with partially or completely people's glycosylation pattern (see Gerngross, T.U., Nat.Biotech.22 (2004) 1409-1414; And Li, H. etc., Nat.Biotech.24 (2006) 210-215).

Be suitable for the host cell of expression glycosylated antibodies also derived from multicellular organism (invertebrates and vertebrates).The example of invertebral zooblast comprises plant and insect cell.Identify many can being combined with insect cell, covet the baculovirus strain of noctuid (Spodopterafrugiperda) cell in particular for transfection meadow.

Plant cell cultures also can be used as host (see such as US5,959,177, US6,040,498, US6,420,548, US7,125,978 and US6,417,429 (describe the PLANTIBODIES being used for producing antibody in transgenic plant ^tMtechnology).

Vertebrate cells also can be used as host.Such as, the mammal cell line adapting to suspension growth can be used.Other examples of useful mammalian host cell line are monkey kidney CV1 clone (COS-7) that SV40 transforms; Human embryonic kidney cell line's (being described in such as Graham, F.L. etc., the HEK293 in J.GenVirol.36 (1977) 59-74 or 293 cells); Baby hamster kidney cell (BHK); Mouse sertoli cells (being described in the TM4 cell in such as Mather, J.P., Biol.Reprod.23 (1980) 243-252); Monkey-kidney cells (CV1); African green monkey kidney cell (VERO-76); Human cervical carcinoma cell (HELA); Madin-Darby canine kidney(cell line) (MDCK); Buffalo rat hepatocytes (BRL3A); Human pneumonocyte (W138); Human liver cell (HepG2); Mouse mammary tumor (MMT060562); Be described in such as Mather, J.P. etc., the TRI cell in AnnalsN.Y.Acad.Sci.383 (1982) 44-68; MRC5 cell; And FS4 cell.Other useful mammal cell lines comprise Chinese hamster ovary (CHO) cell, comprise DHFR ^-chinese hamster ovary celI (Urlaub, G. etc., Proc.Natl.Acad.Sci.USA77 (1980) 4216-4220); And myeloma cell line, as Y0, NS0 and Sp2/0.The summary being suitable for some mammalian host cell line that antibody produces is shown in such as Yazaki, P. and Wu, A.M., MethodsinMolecularBiology, 248 volumes, Lo, B.K.C. (editor), HumanaPress, Totowa, NJ (2004), 255-268 page.

Assay method

By many measure method known in the art, can identify, screen or characterize physical/chemical properties and/or the biologic activity of antibody provided herein.

In one aspect, such as, by known method as ELISA, Western blotting etc., to its antigen-binding activity of the antibody test such as reported herein.

Immunoconjugates

Present invention provides the immunoconjugates of antibody as reported comprising and put together as chemotherapeutics or medicine, growth inhibitor, toxin (such as, bacterial origin, fungic origin, plant-sourced or zoogenous archon, enzyme activity toxin) or radio isotope with one or more cytotoxic agents herein.

In one embodiment, immunoconjugates is antibody-drug conjugates (ADC), wherein antibody and one or more drug conjugate, described medicine includes but not limited to that class maytenin (maytansinoid) is (see US5,208,020, US5,416,064 and EP0425235B1); Auspicious department of Australia statin department as auspicious in monomethyl Australia statins part DE and DF (MMAE and MMAF) (see US5,635,483 and US5,780,588 and US7,498,298); Aplysiatoxin (dolastatin); Calicheamycin or derivatives thereof (see US5,712,374, US5,714,586, US5,739,116, US5,767,285, US5,770,701, US5,770,710, US5,773,001, and US5,877,296; The people such as Hinman, L.M., CancerRes.53 (1993) 3336-3342; AndLode, H.N.etal., CancerRes.58 (1998) 2925-2928); Anthracycline is as daunorubicin or Dx (people such as Kratz, F., Curr.Med.Chem.13 (2006) 477-523; The people such as Jeffrey, S.C., Bioorg.Med.Chem.Lett.16 (2006) 358-362; The people such as Torgov, M.Y., Bioconjug.Chem.16 (2005) 717-721; Nagy, A.etal., Proc.Natl.Acad.Sci.USA97 (2000) 829-834; The people such as Dubowchik, G.M., Bioorg. & Med.Chem.Letters12 (2002) 1529-1532; The people such as King, H.D., J.Med.Chem.45 (2002) 4336-4343; And US6,630,579)); Methotrexate; Vindesine; Taxan is as Docetaxel, taxol, larotaxel, tesetaxel and ortataxel; Trichothecene; And CC1065.

In another embodiment, immunoconjugates comprises the antibody as described herein puted together with enzyme activity toxin or its fragment, described enzyme activity toxin or its fragment include but not limited to diphtheria toxin A chain, diphtheria toxin without binding activities fragment, exotoxin A chain (from Pseudomonas aeruginosa (Pseudomonasaeruginosa)), ricin A chain, abrin A chain, capsule lotus root toxalbumin A chain, α-sarcin, tung oil tree (Aleuritesfordii) albumen, Dianthus caryophyllus L. toxalbumin, pokeroot (Phytolacaamericana) albumen (PAPI, PAPII and PAP-5), balsam pear (momordicacharantia) arrestin, curcin, crotin, Saponaria officinalis (sapaonariaofficinalis) arrestin, spend more gelonin, NSC-69529 (mitogellin), restrictocin, phenomycin, enomycin and Trichothecenes compounds.

In another embodiment, immunoconjugates comprises the antibody as described herein puted together to be formed with radioactive atom and radiate conjugate.Multiple radio isotope can be used for producing radiation conjugate.Example comprises At ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², P ³², Pb ²¹²with the radio isotope of Lu.When radiating conjugate for detecting, it can comprise the radioactive atom for scintillation method research, such as TC ^99mor I ¹²³, or the spin label of nucleus magnetic resonance (NMR) imaging (also referred to as nuclear magnetic resonance, MRI), as iodo-123, iodine-131, indium-111, fluoro-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Multiple difunctionality protein coupling agents can be used to produce the conjugate of antibody and cytotoxic agent, described difunctionality protein coupling agents is as N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP), 4-(N-maleimidomehyl) hexanaphthene-1-carboxylic acid succinimide ester (SMCC), iminothiolane (IT), bifunctional derivative's (as oneself two imide salt dimethyl phthalates) of polyurethane, active ester (as disuccinimidyl suberate), aldehyde (as glutaraldehyde), two-triazo-compound (as two (p-diazonium salt benzoyl) hexanediamine), two-diazonium salt derivative (as two-(p-diazonium salt benzoyl)-quadrol), vulcabond is (as 2, 6-toluene-2,4-diisocyanate) and double activated fluorine cpd (as 1, 5-bis-fluoro-2, 4-dinitrobenzene).Such as, as people such as Vitetta, E.S., described in Science238 (1987) 1098-1104, ricin immunotoxin can be prepared like that.1-isothiocyanatobenzyl-3-methyl diethylenetriamine pentaacetic acid (MX-DTPA) of carbon-14-mark is the Exemplary chelators for radionuclide and antibody conjugate.See WO94/11026.Joint can be " can cut joint " of promoting that cytotoxic agent discharges in cell.Such as, the joint of sour unstable joint, peptidase-sensitive, photo-labile joint, dimethyl linker or the joint containing disulfide linkage (people such as Chari, R.V., CancerRes.52 (1992) 127-131 can be used; US5,208,020).

Immunoconjugates or ADC conceive in this article clearly, but be not limited to this kind of conjugate adopting linking agent reagent to prepare, described linking agent reagent includes but not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo group-EMCS, sulfo group-GMBS, sulfo group-KMUS, sulfo group-MBS, sulfo group-SIAB, sulfo group-SMCC, with sulfo group-SMPB, with can business obtain SVSB (succinimido-(4-vinyl sulfone(RemzaolHuo Xingranliaohuoxingjituan)) benzoic ether) (such as, available from PPierceBiotechnology, Inc., Rockford, Illinois, the U.S.).

For the method and formulation of diagnosis and detection

In certain embodiments, any one antibody provided herein can be used for detecting its one or more close the existence of associated antigen in biological sample.As used herein, quantitative or qualitative detection contained in term " detection ".In certain embodiments, biological sample comprises cell or tissue.

In one embodiment, the antibody reported for diagnosing or in detection method is provided herein.

In certain embodiments, the antibody of the mark reported is provided herein.Marker includes but not limited to, the marker directly detected or part (as fluorescence, color development, electron dense, chemoluminescence and radioactive marker), and indirect detection is to the part of (such as by enzymatic reaction or interaction of molecules), as enzyme or part.Exemplary indicia thing includes but not limited to radio isotope ³²p, ¹⁴c, ¹²⁵i, ³h and ¹³¹i, fluorophore is as rare earth chelate compound or fluorescein and derivative thereof, rhodamine and derivative thereof, red sulphonyl, Umbelliferone, luciferase such as Fluc and bacteriofluorescein enzyme (US4, 737, 456), luciferin, 2, 3-dihydro naphthyridine diketone, horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase enzymes, glucoamylase, N,O-Diacetylmuramidase, carbohydrate oxidase is glucose oxidase such as, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD), Heterocyclic oxidases is as uriKoxidase and XOD, its with utilize hydrogen peroxide to carry out the enzyme of oxidation dye precursors as HRP, lactoperoxidase or microperoxisome coupling, biotin/avidin, spin label, bacteriophage labels thing, stabilized radical etc.

Pharmaceutical preparation

Prepare the pharmaceutical preparation of the antibody as described herein of freeze-dried preparation or aqueous pharmaceutical form in the following manner: this antibody with required purity is mixed (Remington'sPharmaceuticalSciences the 16th edition with one or more optional pharmaceutically acceptable carrier, Osol, A. write (1980)).Pharmaceutically acceptable carrier generally dosage used and concentration nontoxic to recipient, and to include but not limited to: buffer reagent is as phosphoric acid salt, citric acid and other organic acids; Antioxidant (comprising xitix and methionine(Met)); Sanitas is (as octadecyl benzyl dimethyl ammonium chloride; Hexamethonium chloride; Benzalkonium chloride, Morpan BB; Phenol, butanols or benzylalcohol; Alkyl nipagin esters is as Tegosept M or propyl ester; Catechol; Resorcinol; Hexalin; 3-amylalcohol and meta-cresol); Lower molecular weight (being less than about 10 residues) polypeptide; Protein, as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer is as PVP; Amino acid is as glycine, glutamine, l-asparagine, Histidine, arginine or Methionin; Monose, disaccharides and other carbohydrates comprise glucose, seminose or dextrin; Sequestrant is as EDTA; Sugar is as sucrose, seminose, trehalose or sorbose; Form the gegenion of salt as sodium; Metal composite (such as Zn-protein complex) and/or nonionogenic tenside are as polyoxyethylene glycol (PEG).Exemplary pharmaceutically acceptable carrier herein also comprise interstitial drug dispersion agent as soluble neutral reactive transparent matter acid enzyme glycoprotein (sHASEGP), such as, human soluble PH-20 hyaluronidase glycoprotein, as rhuPH20 ( baxterInternational, Inc.).In US2005/0260186 and US2006/0104968, describe some exemplary sHASEGP and using method, comprise rhuPH20.In one aspect, sHASEGP and one or more extra glycosaminoglycan enzyme such as chondroitinases combine.

Exemplary lyophilized antibodies preparation at US6,267, in 958 describe.Water quality antibody preparation is included in US6,171,586 and WO2006/044908 in those preparations of describing, a rear class preparation comprises Histidine-acetate buffer.

Preparation herein also can need according to the specific adaptations disease for the treatment of and containing more than a kind of activeconstituents, preferably have those activeconstituentss of the complementary activity of not disadvantageous effect mutually.This activeconstituents is to be effective to expect that the amount of object suitably exists.

Activeconstituents can be embedded in the microcapsule such as prepared respectively by condensation technique or interfacial polymerization (such as, Walocel MT 20.000PV microcapsule or gelatin-microcapsule and poly-(methyl methacrylate) microcapsule), in colloidal drug delivery systems (such as, liposome, albumi microspheres, microemulsion, nanoparticle and Nano capsule) or emulsion.This type of technology is Remington'sPharmaceuticalSciences the 16th edition, open during Osol, A. write (1980).

Sustained release formulation can be prepared.The suitable example of sustained release formulation comprises the solid hydrophobic polymers semipermeable matrices containing antibody, and described matrix is in moulded products (such as film or microcapsule) form.

Be ready to use in the preparation used in body normally aseptic.Such as can realize sterility easily by filtering by sterilised membrane filter.

Methods for the treatment of and preparation

Any antibody provided herein may be used in methods for the treatment of.

In one aspect, provide as medicine as antibody reported here.

In certain embodiments, the antibody in methods for the treatment of is provided for.In a this embodiment, the method also comprises at least one additional therapeutic agent using significant quantity to this individuality, such as, and therapeutical agent as described below.People in a preferred embodiment according to " individuality " of any one above embodiment.

In yet another aspect, the invention provides antibody in the purposes manufactured or prepare in medicine.Can be people according to " individuality " of any one embodiment in above embodiment.

In yet another aspect, the invention provides the pharmaceutical preparation comprising any antibody provided herein, such as, in aforementioned any methods for the treatment of.In one embodiment, pharmaceutical preparation comprises any antibody provided herein and pharmaceutically acceptable carrier.In another embodiment, pharmaceutical preparation comprises any antibody provided herein and at least one additional therapeutic agent, such as, as described below.

As antibody reported here can in therapy separately or combinationally use with other promoting agents.Such as, as antibody reported here can be used altogether with at least one additional therapeutic agent.

Antibody (with any extra therapeutical agent) as reported herein can be used by any suitable means, described appropriate means comprises parenteral, in lung and in nose and (if topical therapeutic needs) intralesional use.Parenteral infusions comprises in intramuscular, intravenously, intra-arterial, abdomen or subcutaneous administration.Administration can be undertaken by any suitable approach, and such as, by injection, as intravenously or subcutaneous injection, this part ground depends on whether use is of short duration or long-term.Contemplate various dosage regimen herein, include but not limited to single or repeatedly use at various time point, bolus injection uses and pulse infusion.

Antibody as reported herein will be prepared, determine dosage and use in the mode meeting good medical practice.The factor considered in this case comprises the concrete illness for the treatment of, the concrete Mammals treated, the clinical condition of individual patient, illness reason, send other known factors of the position of drug delivery, application process, the plan of using and medical practitioner.Antibody do not need be used at present preventing or treat illness is discussed one or more medicines together with prepare, but optionally prepare together with them.The significant quantity of this kind of other drug depends on the amount of the antibody existed in said preparation, disease type or therapy and other factors discussed above.These medicines usually to adopt uses with identical route of administration described herein with identical dosage described herein, or use with the dosage described herein of about 1% to 99%, or with empirically/determine any dosage of being suitable for and the use of any approach clinically.

For prevention or disease therapy, optimal dose (when separately or when combinationally using with one or more other additional therapeutic agent) as the antibody reported herein will depend on the type of disease to be treated, the type of antibody, the seriousness of disease and process, whether antibody is used for prevention or therapeutic purpose, the decision of the clinical history of previous therapies, patient and the response of antagonist and attending doctor.By antibody suitably by once or through a series for the treatment of being applied to patient.Depend on type and the seriousness of disease, whether about 1 μ g/kg to 15mg/kg (such as 0.5mg/kg-10mg/kg) antibody can be the initial candidate dosage used to patient, no matter such as used separately by one or more or used by continuous infusion.Depend on factor mentioned above, common every per daily dose may be from about 1 μ g/kg to 100mg/kg or more.For repetitive administration in several days or longer time scope, depend on symptom, treatment usually will continue until occur restraining effect needed for disease symptoms.An exemplary dose of antibody will be within the scope of about 0.05mg/kg to about 10mg/kg.Therefore, one or more dosage (or its arbitrary combination) of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg can be used to patient.This kind of dosage can be used off and on, such as weekly or within every 3 weeks, use (such as thus this patient accepts about 2 to about 20 dosage of antibody or such as about 6 dosage).Can use higher initial loading dose, be one or more comparatively low dosages subsequently.The process of this therapy is easily monitored by routine techniques and assay method.

Be appreciated that, the antibody that any one preparation aforementioned or methods for the treatment of can use the immunoconjugates as reported to substitute as reported herein is herein implemented, or can except using the antibody as reported herein, also use the immunoconjugates as reported to implement herein.

Manufacture thing

Such as report herein another in, provide and a kind of manufacture thing, described manufacture thing contain above-describedly can be used for treating, the material of prevention and/or diagnose medical conditions.This manufacture thing comprises container and on this container or the label combined with it or package insert.Suitable container comprises such as bottle, phial, syringe, intravenous fluids bag etc.Container can from multiple material as glass or plastics be formed.This container contain itself or effectively treat with during another kind of formulation compositions, prevent and/or diagnose medical conditions preparation and aseptic access port (such as this container can be intravenous fluids bag or the phial with the transparent bottle stopper of hypodermic needle) can be had.At least one active substance in preparation is the antibody as reported herein.Label or package insert illustrate that said preparation is used for the treatment of the symptom of selection.And manufacture thing and can comprise (a) first container wherein containing preparation, wherein said preparation comprises the antibody as reported herein; (b) second container wherein containing preparation, wherein said preparation comprises other cytotoxic agents or therapeutical agent.Manufacture thing in this embodiment reported herein and can also comprise the package insert that instruction preparation can be used for treating very pathology.Alternatively or extraly, this manufacture thing also can comprise second (or 3rd) container, and it comprises pharmaceutically acceptable buffer reagent, as bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution and glucose solution.It also can comprise welcome other materials viewed from business and User Perspective, comprises other buffers, thinner, filter, syringe needle and syringe.

Be appreciated that aforementioned arbitrary manufacture thing can comprise the immunoconjugates as reported substituted as the antibody reported herein herein, or outside the antibody such as reported, also can comprise the immunoconjugates as reported herein herein.

III. specific embodiments

1. an IgG class Fc district, comprises first variant Fc district's polypeptide and the second variant Fc district polypeptide,

Wherein

A) the first variant Fc district polypeptide derived from first parent IgG class Fc district's polypeptide and the second variant Fc district polypeptide derived from the second parent IgG class Fc district polypeptide, thus first parent IgG class Fc district's polypeptide and the second parent IgG class Fc district polypeptide identical or different, and

B) different with the second variant Fc district polypeptide in one or more amino-acid residues of the first variant Fc district polypeptide except those amino-acid residues different from the second parent IgG class Fc district polypeptide except wherein the first parent IgG class Fc district polypeptide, and

C) comprise the first variant Fc district polypeptide and the IgG class Fc district of the second variant Fc district polypeptide and there is the avidity to people Fc-acceptor different from the IgG class Fc district of the first parent IgG class Fc district's polypeptide comprised a) and the second parent IgG class Fc district polypeptide a).

2., according to the IgG class Fc district of embodiment 1, wherein people Fc-acceptor is people's neonatal Fc receptor (FcRn) or people Fc γ III acceptor (Fc γ RIII).

3., according to the IgG class Fc district of any one embodiment in embodiment 1 to 2, wherein people Fc-acceptor is the newborn Fc-acceptor of people.

4. according to the IgG class Fc district of any one embodiment in embodiment 1 to 3, wherein compare with the IgG class Fc district of the first parent IgG class Fc district polypeptide comprised a) with the second parent IgG class Fc district polypeptide a), measured by surperficial plasmon resonance (SPR), comprise the avidity increase or minimizing 10% or more of IgG class Fc district to people Fc-acceptor of first variant Fc district's polypeptide and the second variant Fc district polypeptide.

5., according to the IgG class Fc district of any one embodiment in embodiment 1 to 4, in those amino-acid residues that wherein the first parent IgG class Fc district polypeptide is different from the second parent IgG class Fc district polypeptide, at least some amino-acid residue promotes the formation in different dimerization IgG class Fc district.

6. according to the IgG class Fc district of any one embodiment in embodiment 1 to 5, wherein

I) the first parent IgG class Fc district polypeptide is selected from

-human IgG1 Fc district polypeptide,

-human IgG2 Fc district polypeptide,

-human IgG 3Fc district polypeptide,

-human IgG 4Fc district polypeptide,

-have sudden change L234A, L235A human IgG1 Fc district polypeptide,

-have sudden change Y349C, T366S, L368A, Y407V human IgG1 Fc district polypeptide,

-have sudden change S354C, T366S, L368A, Y407V human IgG1 Fc district polypeptide,

-have sudden change L234A, L235A, Y349C, T366S, L368A, Y407V human IgG1 Fc district polypeptide,

-have sudden change L234A, L235A, S354C, T366S, L368A, Y407V human IgG1 Fc district polypeptide,

-have sudden change P329G human IgG1 Fc district polypeptide,

-have sudden change L234A, L235A, P329G human IgG1 Fc district polypeptide,

-have sudden change P329G, Y349C, T366S, L368A, Y407V human IgG1 Fc district polypeptide,

-have sudden change P329G, S354C, T366S, L368A, Y407V human IgG1 Fc district polypeptide,

-have sudden change L234A, L235A, P329G, Y349C, T366S, L368A, Y407V human IgG1 Fc district polypeptide,

-have sudden change L234A, L235A, P329G, S354C, T366S, L368A, Y407V human IgG1 Fc district polypeptide,

-have sudden change S228P, L235E human IgG 4Fc district polypeptide,

-have sudden change S228P, L235E, P329G human IgG 4Fc district polypeptide,

-have sudden change Y349C, T366S, L368A, Y407V human IgG 4Fc district polypeptide,

-have sudden change S354C, T366S, L368A, Y407V human IgG 4Fc district polypeptide,

-have sudden change S228P, L235E, Y349C, T366S, L368A, Y407V human IgG 4Fc district polypeptide,

-have sudden change S228P, L235E, S354C, T366S, L368A, Y407V human IgG 4Fc district polypeptide,

-have sudden change P329G human IgG 4Fc district polypeptide,

-have sudden change P329G, Y349C, T366S, L368A, Y407V human IgG 4Fc district polypeptide,

-have sudden change P329G, S354C, T366S, L368A, Y407V human IgG 4Fc district polypeptide,

-have sudden change S228P, L235E, P329G, Y349C, T366S, L368A, Y407V human IgG 4Fc district polypeptide,

-have sudden change S228P, L235E, P329G, S354C, T366S, L368A, Y407V human IgG 4Fc district polypeptide,

-there is the sudden change human IgG1 of K392D, IgG2 or IgG4, and

-have sudden change N392D human IgG 3,

And

Ii) the second parent IgG class Fc district polypeptide is selected from

-human IgG1 Fc district polypeptide,

-human IgG2 Fc district polypeptide,

-human IgG 3Fc district polypeptide,

-human IgG 4Fc district polypeptide,

-have sudden change L234A, L235A human IgG1 Fc district polypeptide,

-have sudden change S354C, T366W human IgG1 Fc district polypeptide,

-have sudden change Y349C, T366W human IgG1 Fc district polypeptide,

-have sudden change L234A, L235A, S354C, T366W human IgG1 Fc district polypeptide,

-have sudden change L234A, L235A, Y349C, T366W human IgG1 Fc district polypeptide,

-have sudden change P329G human IgG1 Fc district polypeptide,

-have sudden change L234A, L235A, P329G human IgG1 Fc district polypeptide,

-have sudden change P329G, S354C, T366W human IgG1 Fc district polypeptide,

-have sudden change P329G, Y349C, T366W human IgG1 Fc district polypeptide,

-have sudden change L234A, L235A, P329G, S354C, T366W human IgG1 Fc district polypeptide,

-have sudden change L234A, L235A, P329G, Y349C, T366W human IgG1 Fc district polypeptide,

-have sudden change S228P, L235E human IgG 4Fc district polypeptide,

-have sudden change S228P, L235E, P329G human IgG 4Fc district polypeptide,

-have sudden change S354C, T366W human IgG 4Fc district polypeptide,

-have sudden change Y349C, T366W human IgG 4Fc district polypeptide,

-have sudden change S228P, L235E, S354C, T366W human IgG 4Fc district polypeptide,

-have sudden change S228P, L235E, Y349C, T366W human IgG 4Fc district polypeptide,

-have sudden change P329G human IgG 4Fc district polypeptide,

-have sudden change P329G, S354C, T366W human IgG 4Fc district polypeptide,

-have sudden change P329G, Y349C, T366W human IgG 4Fc district polypeptide,

-have sudden change S228P, L235E, P329G, S354C, T366W human IgG 4Fc district polypeptide,

-have sudden change S228P, L235E, P329G, Y349C, T366W human IgG 4Fc district polypeptide,

-have sudden change D399K, D356K and/or E357K human IgG1, and

-there is the sudden change human IgG2 of D399K, E356K and/or E357K, IgG3 or IgG4.

7. according to the IgG class Fc district of any one embodiment in embodiment 1 to 6, wherein

I) the first parent IgG class Fc district polypeptide have be selected from SEQIDNO:60,61,62,63,64,65,67,69,70,71,73,75,76,78,80,81, the aminoacid sequence of 82 and 84,

And

Ii) the second parent IgG class Fc district polypeptide have be selected from SEQIDNO:60,61,62,63,64,66,68,69,70,72,74,75,76,77,79,81, the aminoacid sequence of 83 and 85.

8. according to the IgG class Fc district of any one embodiment in embodiment 1 to 7, wherein

I) the first parent IgG class Fc district polypeptide is human IgG1 Fc district's polypeptide and the second parent IgG class Fc district polypeptide is human IgG1 Fc district polypeptide, or

Ii) the first parent IgG class Fc district polypeptide has sudden change human IgG1 Fc district's polypeptide of L234A, L235A and the second parent IgG class Fc district polypeptide is the human IgG1 Fc district polypeptide with sudden change L234A, L235A, or

Iii) the first parent IgG class Fc district polypeptide has sudden change human IgG1 Fc district's polypeptide of L234A, L235A, P329G and the second parent IgG class Fc district polypeptide is the human IgG1 Fc district polypeptide with sudden change L234A, L235A, P329G, or

Iv) the first parent IgG class Fc district polypeptide has sudden change human IgG1 Fc district's polypeptide of L234A, L235A, S354C, T366W and the second parent IgG class Fc district polypeptide is the human IgG1 Fc district polypeptide with sudden change L234A, L235A, Y349C, T366S, L368A, Y407V, or

V) the first parent IgG class Fc district polypeptide has sudden change human IgG1 Fc district's polypeptide of L234A, L235A, P329G, S354C, T366W and the second parent IgG class Fc district polypeptide is the human IgG1 Fc district polypeptide with sudden change L234A, L235A, P329G, Y349C, T366S, L368A, Y407V, or

Vi) the first parent IgG class Fc district polypeptide is human IgG 4Fc district's polypeptide and the second parent IgG class Fc district polypeptide is human IgG 4Fc district polypeptide, or

Vii) the first parent IgG class Fc district polypeptide has sudden change human IgG 4Fc district's polypeptide of S228P, L235E and the second parent IgG class Fc district polypeptide is the human IgG 4Fc district polypeptide with sudden change S228P, L235E, or

Viii) the first parent IgG class Fc district polypeptide has sudden change human IgG 4Fc district's polypeptide of S228P, L235E, P329G and the second parent IgG class Fc district polypeptide is the human IgG 4Fc district polypeptide with sudden change S228P, L235E, P329G, or

Ix) the first parent IgG class Fc district polypeptide has sudden change human IgG 4Fc district's polypeptide of S228P, L235E, S354C, T366W and the second parent IgG class Fc district polypeptide is the human IgG 4Fc district polypeptide with sudden change S228P, L235E, Y349C, T366S, L368A, Y407V, or

X) the first parent IgG class Fc district polypeptide has sudden change human IgG 4Fc district's polypeptide of S228P, L235E, P329G, S354C, T366W and the second parent IgG class Fc district polypeptide is the human IgG 4Fc district polypeptide with sudden change S228P, L235E, P329G, Y349C, T366S, L368A, Y407V.

9. according to the IgG class Fc district of any one embodiment in embodiment 1 to 5 and 7, wherein

I) the first parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:60 and the second parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:60, or

Ii) the first parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:64 and the second parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:64, or

Iii) the first parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:70 and the second parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:70, or

Iv) the first parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:68 and the second parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:67, or

V) the first parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:74 and the second parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:73, or

Vi) the first parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:63 and the second parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:63, or

Vii) the first parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:75 and the second parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:75, or

Viii) the first parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:76 and the second parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:76, or

Ix) the first parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:79 and the second parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:80, or

X) the first parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:85 and the second parent IgG class Fc district polypeptide has the aminoacid sequence of SEQIDNO:84.

10. according to the IgG class Fc district of any one embodiment in embodiment 1 to 9, wherein different with the second variant Fc district polypeptide in 1 to 8 amino-acid residue of the first variant Fc district polypeptide except those amino-acid residues different from the second parent IgG class Fc district polypeptide except wherein the first parent IgG class Fc district polypeptide.

11. according to the IgG class Fc district of any one embodiment in embodiment 1 to 10, wherein different with the second variant Fc district polypeptide in 1 to 6 amino-acid residue of the first variant Fc district polypeptide except those amino-acid residues different from the second parent IgG class Fc district polypeptide except wherein the first parent IgG class Fc district polypeptide.

12. according to the IgG class Fc district of any one embodiment in embodiment 1 to 11, wherein different with the second variant Fc district polypeptide in 1 to 3 amino-acid residue of the first variant Fc district polypeptide except those amino-acid residues different from the second parent IgG class Fc district polypeptide except wherein the first parent IgG class Fc district polypeptide.

13. according to the IgG class Fc district of any one embodiment in embodiment 1 to 12, wherein the first variant Fc district polypeptide is the 228th, 234, 235, 236, 237, 238, 239, 248, 250, 251, 252, 253, 254, 255, 256, 257, 265, 266, 267, 268, 269, 270, 272, 285, 288, 290, 291, 297, 298, 299, 307, 308, 309, 310, 311, 314, 327, 328, 329, 330, 331, 332, 385, 387, 428, 433, 434, 435 with different from the second variant Fc district polypeptide in one or more amino-acid residues at 436 (according to KabatEUindex number system numbering) places.

14. according to the IgG class Fc district of any one embodiment in embodiment 1 to 2 and 4 to 13, and wherein the first variant Fc district polypeptide is different from the second variant Fc district polypeptide in the 228th, 234,235,236,237,238,239,253,254,265,266,267,268,269,270,288,297,298,299,307,311,327,328,329,330,331,332,434 with one or more amino-acid residues at 435 (according to KabatEUindex number system numbering) places.

15. according to the IgG class Fc district of any one embodiment in embodiment 1 to 2 and 4 to 14, and wherein the first variant Fc district polypeptide is different from the second variant Fc district polypeptide in the 233rd, 236,265,297,329 with one or more amino-acid residues at 331 places.

16. according to the IgG class Fc district of embodiment 15, wherein the first variant Fc district polypeptide because of one or more amino acid change E233P, Δ G236, D265A, N297A, N297D, P329A and P331S different from the second variant Fc district polypeptide.

17. according to the IgG class Fc district of any one embodiment in embodiment 1 to 3 and 5 to 13, and wherein the first variant Fc district polypeptide is different from the second variant Fc district polypeptide in the 248th, 250,251,252,253,254,255,256,257,272,285,288,290,291,308,309,310,311,314,385,386,387,428,432,433,434,435 with one or more amino-acid residues at 436 places.

18. according to the IgG class Fc district of any one embodiment in embodiment 1 to 3 and 5 to 17, and wherein the first variant Fc district polypeptide is different with the second variant Fc district polypeptide from the one or more amino-acid residues located at the 251st, 253,310,314,432,433,435 and 436.

19. according to the IgG class Fc district of any one embodiment in embodiment 1 to 3 and 5 to 18, and wherein a Fc district polypeptide i) organizes I253A because being selected from, H310A and H435A, or ii) organize H310A, H433A and Y436A, or iii) organize L251D, L314D and L432D, or iv) organize L251S, one or two sudden change of L314S with L432S (according to KabatEUindex number system numbering) is different from the 2nd Fc district polypeptide, and the 2nd Fc district polypeptide is because being selected from sudden change L251D, L251S, I253A, H310A, L314D, L314S, L432D, L432S, H433A, one or two sudden change of H435A with Y436A (according to KabatEUindex number system numbering) is different from a Fc district polypeptide, thus the whole sudden changes when merging consideration in the first and second Fc district polypeptide cause sudden change i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S is contained in this Fc district.

20. according to the IgG class Fc district of any one embodiment in embodiment 1 to 3 and 5 to 18, wherein a Fc district polypeptide i) organizes I253A because being selected from, H310A and H435A, or ii) organize H310A, one or two sudden change of H433A with Y436A (according to KabatEUindex number system numbering) is different from the 2nd Fc district polypeptide, and the 2nd Fc district polypeptide is because being selected from sudden change I253A, H310A, H433A, one or two sudden change of H435A with Y436A (according to KabatEUindex number system numbering) is different from a Fc district polypeptide, thus the whole sudden changes when considering in the lump in the first and second Fc district polypeptide cause sudden change i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A is contained in this Fc district.

21. according to the IgG class Fc district of any one embodiment in embodiment 1 to 3 and 5 to 18, sudden change I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D or L251S/L314S/L432S or its combination (according to KabatEUindex number system numbering) is comprised in Qi Fc district, thus i) all sudden change all first or the 2nd in Fc district polypeptide, or ii) one or two sudden change in a Fc district polypeptide and one or two sudden change in the 2nd Fc district polypeptide, thus the whole sudden changes when considering in the lump in the first and second Fc district polypeptide cause sudden change i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S is contained in this Fc district.

22. according to the IgG class Fc district of any one embodiment in embodiment 1 to 3 and 5 to 18, sudden change I253A/H310A/H435A or H310A/H433A/Y436A or its combination (according to KabatEUindex number system numbering) is comprised in Qi Fc district, thus i) all sudden change all first or the 2nd in Fc district polypeptide, or ii) one or two sudden change in a Fc district polypeptide and one or two sudden change in the 2nd Fc district polypeptide, thus the whole sudden changes when considering in the lump in the first and second Fc district polypeptide cause sudden change i) I253A, H310A and H435A, or ii) H310A, H433A and Y436A is contained in this Fc district.

23. according to the IgG class Fc district of any one embodiment in embodiment 1 to 3 and 5 to 22, wherein compare with the IgG class Fc district of the first parent IgG class Fc district polypeptide comprised a) with the second parent IgG class Fc district polypeptide a), described IG class Fc district has the keying action to SP of reduction.

24. according to the IgG class Fc district of any one embodiment in embodiment 1 to 3 and 5 to 17, sudden change M252Y/S254T/T256E (according to KabatEUindex number system numbering) is comprised in Qi Fc district, thus i) all sudden change all first or the 2nd in Fc district polypeptide, or ii) one or two sudden change in a Fc district polypeptide and one or two sudden change in the 2nd Fc district polypeptide, thus the whole sudden changes when considering in the lump in the first and second Fc district polypeptide cause sudden change M252Y/S254T/T256E be contained in this IgG class Fc district.

25. antibody, it comprises the IgG class Fc district according to any one embodiment in embodiment 1 to 24.

26. according to the antibody of embodiment 25, and wherein antibody is monoclonal antibody.

27. according to the antibody of any one embodiment in embodiment 25 to 26, and wherein antibody is people's antibody, humanized antibody or chimeric antibody.

28. according to the antibody of any one embodiment in embodiment 25 to 27, and wherein antibody is bi-specific antibody.

29. according to the antibody of any one embodiment in embodiment 25 to 28, and wherein antibody is bivalent antibody.

30. according to the antibody of any one embodiment in embodiment 25 to 29, wherein antibody is the dual specific bivalent antibody that FcRn keying action is eliminated, and it comprises and the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding.

The dual specific bivalent antibody that 31.FcRn keying action is eliminated, it comprises and the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding,

Wherein

In heavy-chain variable domains, the CDR1H district of the CDR3H district of SEQIDNO:14, the CDR2H district of SEQIDNO:15 and SEQIDNO:16 α) is comprised with the first antigen binding site of VEGF specific binding, and in light variable domains, comprise the CDR1L district of the CDR3L district of SEQIDNO:17, the CDR2L district of SEQIDNO:18 and SEQIDNO:19, and

In heavy-chain variable domains, the CDR1H district of the CDR3H district of SEQIDNO:22, the CDR2H district of SEQIDNO:23 and SEQIDNO:24 β) is comprised with the second antigen binding site of ANG-2 specific binding, and in light variable domains, comprise the CDR1L district of the CDR3L district of SEQIDNO:25, the CDR2L district of SEQIDNO:26 and SEQIDNO:27, and

γ) bi-specific antibody comprises the IgG class Fc district according to any one embodiment in embodiment 1 to 24.

32. according to the bi-specific antibody of embodiment 31, wherein

Aminoacid sequence α) comprising SEQIDNO:20 with the first antigen binding site of VEGF specific binding as heavy-chain variable domains VH and the aminoacid sequence comprising SEQIDNO:21 as light variable domains VL, and

Aminoacid sequence β) comprising SEQIDNO:28 with the second antigen binding site of ANG-2 specific binding as heavy-chain variable domains VH and the aminoacid sequence comprising SEQIDNO:29 as light variable domains VL, and

The dual specific bivalent antibody that 33.FcRn keying action is eliminated, it comprises with the heavy chain of the first full length antibody of VEGF specific binding and light chain and with the modification heavy chain of the second full length antibody of ANG-2 specific binding with modify light chain, wherein constant domain CL and CH1 replaces mutually, and described dual specific bivalent antibody comprises

α) aminoacid sequence of SEQIDNO:38 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:40 light chain as the first full length antibody, and

β) aminoacid sequence of SEQIDNO:39 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:41 modification light chain as the second full length antibody.

The dual specific bivalent antibody that 34.FcRn keying action is eliminated, it comprises with the heavy chain of the first full length antibody of VEGF specific binding and light chain and with the modification heavy chain of the second full length antibody of ANG-2 specific binding with modify light chain, wherein constant domain CL and CH1 replaces mutually, and described dual specific bivalent antibody comprises

α) aminoacid sequence of SEQIDNO:34 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:36 light chain as the first full length antibody, and

β) aminoacid sequence of SEQIDNO:35 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:37 modification light chain as the second full length antibody.

The dual specific bivalent antibody that 35.FcRn keying action is eliminated, it comprises with the heavy chain of the first full length antibody of VEGF specific binding and light chain and with the modification heavy chain of the second full length antibody of ANG-2 specific binding with modify light chain, wherein constant domain CL and CH1 replaces mutually, and described dual specific bivalent antibody comprises

α) aminoacid sequence of SEQIDNO:42 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:44 light chain as the first full length antibody, and

β) aminoacid sequence of SEQIDNO:43 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:45 modification light chain as the second full length antibody.

The dual specific bivalent antibody that 36.FcRn keying action is eliminated, it comprises with the heavy chain of the first full length antibody of VEGF specific binding and light chain and with the modification heavy chain of the second full length antibody of ANG-2 specific binding with modify light chain, wherein constant domain CL and CH1 replaces mutually, and described dual specific bivalent antibody comprises

α) aminoacid sequence of SEQIDNO:90 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:40 light chain as the first full length antibody, and

β) aminoacid sequence of SEQIDNO:91 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:41 modification light chain as the second full length antibody.

The dual specific bivalent antibody that 37.FcRn keying action is eliminated, it comprises with the heavy chain of the first full length antibody of VEGF specific binding and light chain and with the modification heavy chain of the second full length antibody of ANG-2 specific binding with modify light chain, wherein constant domain CL and CH1 replaces mutually, and described dual specific bivalent antibody comprises

α) aminoacid sequence of SEQIDNO:88 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:36 light chain as the first full length antibody, and

β) aminoacid sequence of SEQIDNO:89 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:37 modification light chain as the second full length antibody.

The dual specific bivalent antibody that 38.FcRn keying action is eliminated, it comprises with the heavy chain of the first full length antibody of VEGF specific binding and light chain and with the modification heavy chain of the second full length antibody of ANG-2 specific binding with modify light chain, wherein constant domain CL and CH1 replaces mutually, and described dual specific bivalent antibody comprises

α) aminoacid sequence of SEQIDNO:92 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:44 light chain as the first full length antibody, and

β) aminoacid sequence of SEQIDNO:93 is as the modification heavy chain of the second full length antibody and the aminoacid sequence of the SEQIDNO:45 modification light chain as the second full length antibody.

The bi-specific antibody that 39.FcRn keying action is eliminated, its comprise with the heavy chain of the first full length antibody of VEGF specific binding and light chain and with the heavy chain of the second full length antibody of ANG-2 specific binding and light chain, wherein the N-terminal of heavy chain is connected with the C-terminal of light chain by peptide linker (peptidiclinker), and described bi-specific antibody comprises

α) aminoacid sequence of SEQIDNO:46 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:48 light chain as the first full length antibody, and

β) aminoacid sequence of SEQIDNO:47 is as the heavy chain of the second full length antibody be connected with the light chain of the second full length antibody by peptide linker.

The bi-specific antibody that 40.FcRn keying action is eliminated, its comprise with the heavy chain of the first full length antibody of VEGF specific binding and light chain and with the heavy chain of the second full length antibody of ANG-2 specific binding and light chain, wherein the N-terminal of heavy chain is connected with the C-terminal of light chain by peptide linker, and described bi-specific antibody comprises

α) aminoacid sequence of SEQIDNO:49 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:51 light chain as the first full length antibody, and

β) aminoacid sequence of SEQIDNO:50 is as the heavy chain of the second full length antibody be connected with the light chain of the second full length antibody by peptide linker.

The bi-specific antibody that 41.FcRn keying action is eliminated, its comprise with the heavy chain of the first full length antibody of VEGF specific binding and light chain and with the heavy chain of the second full length antibody of ANG-2 specific binding and light chain, wherein the N-terminal of heavy chain is connected with the C-terminal of light chain by peptide linker, and described bi-specific antibody comprises

α) aminoacid sequence of SEQIDNO:94 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:48 light chain as the first full length antibody, and

β) aminoacid sequence of SEQIDNO:95 is as the heavy chain of the second full length antibody be connected with the light chain of the second full length antibody by peptide linker.

The bi-specific antibody that 42.FcRn keying action is eliminated, its comprise with the heavy chain of the first full length antibody of VEGF specific binding and light chain and with the heavy chain of the second full length antibody of ANG-2 specific binding and light chain, wherein the N-terminal of heavy chain is connected with the C-terminal of light chain by peptide linker, and described bi-specific antibody comprises

α) aminoacid sequence of SEQIDNO:96 is as the heavy chain of the first full length antibody and the aminoacid sequence of the SEQIDNO:51 light chain as the first full length antibody, and

β) aminoacid sequence of SEQIDNO:97 is as the heavy chain of the second full length antibody be connected with the light chain of the second full length antibody by peptide linker.

43. according to the bi-specific antibody of any one embodiment in embodiment 39 to 42, wherein by introducing disulfide linkage between heavy-chain variable domains position 44 and light variable domains position 100, the heavy chain of antibody variable domains (VH) of the heavy chain of the second full length antibody and light chain and light chain of antibody variable domains (VL) are carried out disulfide-stabilized (numbering according to Kabat).

44. according to the bi-specific antibody of any one embodiment in embodiment 33 to 43, wherein bi-specific antibody comprises sudden change S354C, T366W and in another structural domain of two CH3 structural domains, comprises sudden change Y349C, T366S, L368A, Y407V in one of two CH3 structural domains, or wherein bi-specific antibody comprises sudden change Y349C, T366W and in another structural domain of two CH3 structural domains, comprises sudden change S354C, T366S, L368A, Y407V in one of two CH3 structural domains.

45. according to the bi-specific antibody of any one embodiment in embodiment 33 to 43, wherein bi-specific antibody comprises sudden change S354C in one of two CH3 structural domains, T366W and comprise in another structural domain of two CH3 structural domains sudden change Y349C, T366S, L368A, Y407V, and except sudden change Y349C in CH3 structural domain, T366S, L368A, sudden change R409D is also comprised outside Y407V, K370E and in CH3 structural domain except sudden change S354C, sudden change D399K is also comprised outside T366W, E357K, or wherein bi-specific antibody comprises sudden change Y349C in one of two CH3 structural domains, T366W and comprise in another structural domain of two CH3 structural domains sudden change S354C, T366S, L368A, Y407V, and except sudden change S354C in CH3 structural domain, T366S, L368A, sudden change R409D is also comprised outside Y407V, K370E and in CH3 structural domain except sudden change Y349C, sudden change D399K is also comprised outside T366W, E357K.

46. according to the bi-specific antibody of any one embodiment in embodiment 25 to 45, and wherein antibody has following one or more characteristic:

-with without iii) under described sudden change corresponding bi-specific antibody compared with, show comparatively low-serum-concentration (for mouse FcRn defect but for applying latter 96 hours in vitreum in for the genetically modified mouse of hemizygote of people FcRn), and/or,

-with without iii) under described sudden change corresponding bi-specific antibody compared with, similar (multiple 0.8 to 1.2) concentration is shown (for mouse FcRn defect but for in the genetically modified mouse of hemizygote of people FcRn in full right eye lysate, latter 96 hours are applied in vitreum in right eye), and/or

-display is not combined with people's neonatal Fc receptor, and/or

-display is not combined with SP, and/or

-display is combined with SP.

47.Fc district fusion polypeptide, it comprises the IgG class Fc district according to any one embodiment in embodiment 1 to 24.

48. pharmaceutical preparations, it comprises the antibody according to any one embodiment in embodiment 25 to 46 or the Fc district fusion polypeptide according to embodiment 47.

49. according to the pharmaceutical preparation of embodiment 48, and its pharmaceutical formulations is used for the treatment of Ocular Vessels disease.

50. antibody according to any one embodiment in embodiment 25 to 46 or the Fc district fusion polypeptide according to embodiment 47, it is used as medicine.

51. according to the purposes of embodiment 50, and wherein purposes is used for the treatment of Ocular Vessels disease.

52. antibody according to any one embodiment in embodiment 25 to 46 or the Fc district fusion polypeptide according to embodiment 47 are manufacturing the purposes in medicine.

53. according to the purposes of embodiment 52, and wherein purposes is the medicine for the manufacture of being used for the treatment of Ocular Vessels disease.

54. antibody according to any one embodiment in claim 25 to 46 or the Fc district fusion polypeptide according to embodiment 47, it is used for the treatment of Ocular Vessels disease.

55. treat the method suffering from the patient of Ocular Vessels disease in the following manner: use the antibody according to any one embodiment in embodiment 25 to 46 or the Fc district fusion polypeptide according to embodiment 47 to needing the patient of this treatment.

IV. embodiment

It is below the embodiment as method and formulation reported here.Should be appreciated that in view of generality provided above describes, other embodiments multiple can be implemented.

Although for the object of clear understanding, describe aforementioned invention in detail to illustrate to a certain degree with way of example, these illustrate and example should not be interpreted as limiting as scope reported here.The whole patent quoted herein and the disclosure of scientific literature is complete clearly is by reference incorporated to.

method

electrospray ionization mass spectrometry (ESI-MS)

By adding 0.5 μ LN-glycanase plus (Roche) and sodium phosphate buffer (0.1M, pH7.1) to obtain final sample volume 115 μ L, make protein aliquots containig (50 μ g) de-glycosylation.By mixture 37 DEG C of incubations 18 hours.After this in order to reduce and sex change, the 0.5MTCEP (Pierce) of 60 μ L in 4M guanidine * HCl (Pierce) and 50 μ L8M guanidine * HCl is added.By mixture 37 DEG C of incubations 30 minutes.By size exclusion chromatography (40% acetonitrile, containing 2% formic acid for SepharoseG-25, isoconcentration), by sample desalination.Be equipped with the Q-TOF instrument (maXis, Bruker) upper record ESI mass spectrum (+ve) in nanoESI source (TriVersaNanoMate, Advion).MS optimum configurations is as follows: transfer: funnel RF, 400Vpp; ISCID energy, 0eV; Multipole RF, 400Vpp; Four poles: ion energy, 4.0eV; Inferior quality, 600m/z; Source: dry gas, 8L/ minute; Dry gas temperature, 160 DEG C; Collision cell: impact energy, 10eV; Collision RF:2000Vpp; Ion water cooler: ion water cooler RF, 300Vpp; Transfer time: 120 μ s; Store before pulse, 10 μ s; Sweep limit m/z600 to 2000.In order to evaluating data, use and own develop software (MassAnalyzer).

fcRn surface plasmon resonance (SPR) analyzes

Use BIAcoreT100 instrument (BIAcoreAB, Uppsala, Sweden), by surperficial plasmon resonance (SPR) technical Analysis wild-type antibodies and mutant to the binding characteristic of FcRn.This system is through fully setting up for studying interaction of molecules.It allows continuous real-time monitoring part/analyte keying action, and therefore arranges lower mensuration kinetic parameter in multiple analysis.SPR-technology is based on the specific refractory power measured near gold-plated biosensor chip surface.The change of specific refractory power represent analyte that this is injected in fixed ligand and solution on the surface interact caused by quality change.If the fixed ligand of molecule on surface is combined, then quality increases, when dissociating, then and Mass lost.In current assay method, by amine coupling method, FcRn acceptor is fixed on BIAcoreCM5-biologic sensor chip (GEHealthcareBioscience, Uppsala, Sweden) to 400 response unit (RU) levels.This assay method is implemented in room temperature, with PBS, 0.05%Tween-20 ^tMpH6.0 (GEHealthcareBioscience) is as running buffer and dilution buffer.200nM antibody samples is injected with flow velocity 50 μ in room temperature for L/ minute.Binding time is 180 seconds, dissociates 360 seconds mutually consuming time.By of short duration injection HBS-P, pH8.0, realize the regeneration of chip surface.By comparing injection latter 180 seconds and the injection biologically signal height of latter 300 seconds, carry out the evaluation of SPR-data.Relevant parameter is RU highest level (injecting latter 180 seconds) and stability in late period (injection terminates latter 300 seconds).

albumin A surface plasmon resonance (SPR) is analyzed

This assay method is based on surperficial plasmon resonance light spectrometry.Albumin A is fixed on the surface of surface plasmon resonance biosensor.Once be entered by Sample Injection in the flow cell of SPR spectrometer, it and immobilization albumin A form mixture, cause censorchip surface to improve quality increase, and therefore cause higher response (as 1RU is defined as 1pg/mm ²).After this, by sample dissolution-protein A complexes, reg sensor chip.Subsequently response unit (RU) evaluation signal height and behavior of dissociating are pressed to the response obtained.

By using the amine coupling reagent kit of GEHealthcare, about 3,500 albumin As (20 μ g/ml) of replying unit (RU) are coupled on CM5 chip (GEHealthcare) at pH4.0.

Sample and system buffer liquid are HBS-P+ (0.01MHEPES, 0.15MNaCl, 0.005% tensio-active agent P20, pH7.4 of sterile filtration).Flow cell temperature is set to 25 DEG C and sample area chambers temp is set to 12 DEG C.This system running buffer causes.Subsequently, by 5nM sample construct solution with flow velocity 30 μ injection in L/ minute 120 seconds, be 300 seconds phases of dissociating subsequently.Subsequently, within L/ minute, glycine-HClpH1.5 was injected for a long time by twice 30 seconds with flow velocity 30 μ, reg sensor chip surface.The each sample of triplicate measurement.

bi-specific antibody and their respective sequence

" there is sudden change IHH-AAA " as the term is employed herein and refer to sudden change I253A (Ile253Ala) in the constant heavy district (EUindex according to Kabat numbers) of IgG1 or IgG4 subclass, the combination of H310A (His310Ala) and H435A (His435Ala), " there is sudden change HHY-AAA " as the term is employed herein and refer to sudden change H310A (His310Ala) in the constant heavy district (EUindex according to Kabat numbers) of IgG1 or IgG4 subclass, the combination of H433A (His433Ala) and Y436A (Tyr436Ala), " there is sudden change P329GLALA " as the term is employed herein and refer to sudden change L234A (Leu234Ala) in the constant heavy district (EUindex according to Kabat numbers) of IgG1 subclass, the combination of L235A (Leu235Ala) and P329G (Pro329Gly), and " there is sudden change SPLE " as the term is employed herein and refer to the combination of sudden change S228P (Ser228Pro) and L235E (Leu235Glu) in the constant heavy district (EUindex according to Kabat numbers) of IgG4 subclass.

general introduction

About the general information of the nucleotide sequence of human normal immunoglobulin light chain and heavy chain at Kabat, E.A. people is waited, SequencesofProteinsofImmunologicalInterest, 5th edition, PublicHealthService, NationalInstitutesofHealth, Bethesda, MD provide in (1991).Number according to EU and mention amino-acid residue (people such as Edelman, G.M., Proc.Natl.Acad.Sci.USA63 (1969) 78-85 of antibody chain; The people such as Kabat, E.A., SequencesofProteinsofImmunologicalInterest, the 5th edition, PublicHealthService, NationalInstitutesofHealth, Bethesda, MD (1991)).

recombinant DNA technology

As people such as Sambrook, J., MolecularCloning:Alaboratorymanual; Standard method described in ColdSpringHarborLaboratoryPress, ColdSpringHarbor, NewYork (1989) is used for operating DNA.Specification sheets according to manufacturers uses molecular biology reagents.

gene chemical synthesis

Goal gene section is ordered at Geneart (Regensburg Kreis, Germany) according to the specification sheets provided.

determined dna sequence

DNA sequence dna is measured by the double-strand sequencing of carrying out at MediGenomixGmbH (Martinsried, Germany) or SequiServeGmbH (Vaterstetten, Germany).

dNA and protein sequence analysis and sequence data management

GCG (Genetics Computer group (GeneticsComputerGroup), Madison, Wisconsin) software package the 10.2nd edition and the VectorNTIAdvance of Infomax be set with 8.0 editions generate for sequence, mapping, analyze, annotation and diagram.

expression vector

In order to express described antibody, use based on adopting or do not adopt the cDNA systematism of CMV-intron A promotor or based on adopting the expression plasmid of genomic organization of CMV promoter for transient expression (such as in HEK293-F cell).

The transcription unit of antibody gene is made up of following element:

-at the single restriction site of 5' end,

-from the early stage immediately enhanser of human cytomegalic inclusion disease virus and promotor,

-in the organized situation of cDNA intron A sequence,

The 5' non-translational region of-human immunoglobulin gene,

The nucleic acid of-encode immunoglobulin heavy signal sequence,

The nucleic acid of-encoding human antibody chains (wild-type or there is Domain swapping), described nucleic acid is as cDNA or in genomic organization, have immunoglobulin (Ig) exon: intron systematism,

-there is 3 ' non-translational region of polyadenylation signal sequence, and

-at the single restriction site of 3' end.

Except antibody expression box, plasmid also contains:

-the replication orgin that allows this plasmid to copy in intestinal bacteria,

-in intestinal bacteria, give the β-lactamase gene of amicillin resistance, and

-as the dihydrofolate reductase gene from house mouse (Musmusculus) of selective marker in eukaryotic cell.

The nucleic acid of encoding antibody chain is generated by PCR and/or gene synthesis and passes through to connect corresponding nucleic acid segment, such as, utilize the single restriction enzyme sites in respective carrier to connect, by known recombination method and technology assembling.By the nucleotide sequence of DNA sequencing checking subclone.In order to transient transfection, prepare plasmid from the culture of Escherichia coli (NucleobondAX, Macherey-Nagel) transformed, obtained relatively large plasmid.

cell culture technology

Use as CurrentProtocolsinCellBiology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, and Yamada J., K.M. (write), JohnWiley & Sons, the standard cell culture techniques described in Inc..

As described below, by transient cotransfection expression plasmid separately in the HEK293-F cell grown according to suspended pattern, express bi-specific antibody.

embodiment 1

Expression and purification

transient transfection in HEK293-F system

Use HEK293-F system (Invitrogen), according to the specification sheets of manufacturers, by with respective plasmid (plasmid of the heavy chain of such as encoding heavy chain and modification and the light chain of corresponding light chain and modification) transient transfection, produce monospecific and bi-specific antibody.In brief, by serum-free FreeStyle in shaking flask or in stirred-tank fermenter ^tM293 express in substratum (Invitrogen) with HEK293-F cell (Invitrogen) respective expression plasmid and the 293fectin of suspended pattern growth ^tMor the mixture transfection of fectin (Invitrogen).For 2L shaking flask (Corning), by HEK293-F cell with density 1*10 ⁶individual cell/mL to be seeded in 600mL and with 120 revs/min, 8%CO ₂incubation.After this day, by about 1.5*10 ⁶cell about below 42mL mixture transfection of individual cell/mL cell density: A) mixture of 20mLOpti-MEM (Invitrogen) and the difference encoding heavy chain of 600 μ g equimolar ratio rates or total plasmid DNA (1 μ g/mL) of modified heavy chain and corresponding light chain and B) mixture of 20mlOpti-MEM and 1.2mL293fectin or fectin (2 μ L/mL).During fermentation process according to glucose utilization, add glucose solution.Within 5-10 days, gather in the crops supernatant liquor containing circulating antibody afterwards and by antibody directly from supernatant liquor purifying or supernatant liquor is freezing and store.

purifying

By using MabSelectSure-Sepharose ^tM(for without-IHH-AAA mutant) (GEHealthcare, Sweden) or KappaSelect-Agarose (for IHH-AAA mutant) (GEHealthcare, Sweden) affinity chromatography, use butyl-Sepharose (GEHealthcare, Sweden) hydrophobic interaction chromatography and Superdex200 size exclusion (GEHealthcare, Sweden) chromatography, from cell culture supernatant purifying bispecific antibody.

In brief, with PBS damping fluid (10mMNa ₂hPO ₄, 1mMKH ₂pO ₄137mMNaCl and 2.7mMKCl, pH7.4) the MabSelectSuRe resin that balances (without-IHH-AAA sudden change and wild-type antibodies) is caught the cell culture supernatant of sterile filtration, with equilibration buffer solution and with 25mMpH3.0 Trisodium Citrate wash-out.IHH-AAA mutant caught by the KappaSelect resin balanced with 25mMTris, 50mMNaCl, pH7.2, with equilibration buffer solution and with 25mMpH2.9 Trisodium Citrate wash-out.Merged by the antibody fractions of wash-out and use 2MTris, pH9.0 neutralizes.Use second acid for adjusting pH to pH5.0 by adding 1.6M ammoniumsulphate soln to final concentration 0.8M ammonium sulfate, antibody consolidated material is ready for hydrophobic interaction chromatography.Use 35mM sodium acetate, 0.8M ammonium sulfate, antibody is applied to resin, with equilibration buffer solution and with linear gradient elution to 35mM sodium acetate pH5.0 after balancing butyl-Sepharose resin by pH5.0.Fraction containing (monospecific or dual specific) antibody is merged and uses with 20mM Histidine, 140mMNaCl, Superdex20026/60GL (GEHealthcare, the Sweden) post of pH6.0 balance, further by size exclusion chromatography purifying.Fraction containing (monospecific or dual specific) antibody is merged, uses Vivaspin ultra-filtration equipment (SartoriusStedimBiotechS.A., France) be concentrated into the concentration of requirement and be stored in-80 DEG C.

Table: the productive rate of dual specific <VEGF-ANG-2> antibody

Use microfluid Labchip technology (CaliperLifeScience, the U.S.) by CE-SDS purity assay and antibody integrity after often kind of purification step.According to the specification sheets of manufacturers, use HT protein expression Reagent Kit, prepare the protein soln that 5 μ L analyze for CE-SDS, and use HT protein expression chip, LabChipGXII system is analyzed.Use LabChipGX software analysis data.

Table: determine to remove typical by-product by different order purification steps by CE-SDS

Use the analytical size-exclusion column of Superdex200 (GEHealthcare, Sweden) at 2xPBS (20mMNa at 25 DEG C ₂hPO ₄, 2mMKH ₂pO ₄, 274mMNaCl and 5.4mMKCl, pH7.4) and in running buffer, analyzed the aggregation content of antibody samples by high-performance SEC.25 μ g protein to be expelled on post with flow velocity 0.75mL/ minute and through 50 minutes Isocratic clutions.

Similarly, <VEGF-ANG-2> bi-specific antibody VEGFang2-0012 and VEGFang2-0201 with following productive rate purifying is prepared:

In addition, can prepare similarly and purifying have IHH-AAA suddenly change and have SPLE suddenly change <VEGF-ANG-2> bi-specific antibody <VEGF-ANG-2>CrossMAbIgG4 (SEQIDNO:42, SEQIDNO:43, SEQIDNO:44, SEQIDNO:45), there is the <VEGF-ANG-2>OAscFabIgG1 (SEQIDNO:46 of IHH-AAA sudden change, SEQIDNO:47, SEQIDNO:48), there is IHH-AAA suddenly change and the <VEGF-ANG-2>OAscFabIgG4 (SEQIDNO:49 with SPLE sudden change, SEQIDNO:50, SEQIDNO:51), there is the <VEGF-ANG-2>CrossMabIgG1 (SEQIDNO:90 of HHY-AAA sudden change and P329GLALA sudden change, SEQIDNO:91, SEQIDNO:40, SEQIDNO:41), there is the <VEGF-ANG-2>CrossMabIgG4 (SEQIDNO:92 of HHY-AAA sudden change and SPLE sudden change, SEQIDNO:93, SEQIDNO:44, SEQIDNO:45), there is the <VEGF-ANG-2>OAscFabIgG1 (SEQIDNO:94 of HHY-AAA sudden change, SEQIDNO:95, SEQIDNO:48) and have HHY-AAA sudden change and SPLE suddenly change <VEGF-ANG-2>OAscFabIgG4 (SEQIDNO:96, SEQIDNO:97, SEQIDNO:51).

embodiment 2

Analytics and developability

viscosity measurement based on small-scale DLS:

Viscosity measurement is carried out substantially as described in people such as (, AnalyticalBiochemistry399 (2009) 141-143) He, F..In brief, by sample at 200mM argininosuccinate hydrochlorate, in pH5.5, be concentrated to multiple proteins concentration, add polystyrene latex pearl (300nm diameter) and polysorbate20 (0.02%v/v) afterwards.By sample, to be transferred to optics 384 hole through 0.4 μm of filter plate dull and stereotyped and cover with paraffin oil by centrifugal.The apparent diameter of dynamic light scattering determination latex bead is passed through at 25 DEG C.The viscosity of solution may be calculated η=η 0 (rh/rh, 0) (η: viscosity; η 0: the viscosity of water; Rh: the apparent hydrodynamic radius of latex bead; Rh, 0: the hydrodynamic radius of latex bead in water).

In order to allow at the more various sample of same concentrations, viscosity-concentration data Mooney equation (equation 1) (Mooney, M., Colloid.Sci., 6 (1951) 162-170; Monkos, K., Biochem.Biophys.Acta304 (1997) 1339) matching interpolative data accordingly.

η = η_{0} \exp (\frac{S Φ}{1 - K Φ})

Equation 1

(S: the hydrodynamic interaction parameter of protein; K: self-crowding factor; Φ: the volume fraction of the protein of dissolving)

Show result in Fig. 2: compared with the VEGFang2-0015 suddenlyd change without IHH-AAA in Fc district, the VEGFang2-0016 in Fc district with IHH-AAA sudden change all shows lower viscosity at whole measuring tempeature.

dLS assembles starting temperature

By sample with the concentration of 1mg/mL at 20mM Histidine/L-Histidine hydrochloride, prepare in 140mMNaCl, pH6.0, to be transferred to optics 384 hole through 0.4 μm of filter plate dull and stereotyped and cover with paraffin oil by centrifugal.Sample is heated to while 80 DEG C with the speed of 0.05 DEG C/min from 25 DEG C, by dynamic light scattering method replicate measurement hydrodynamic radius.Assemble starting temperature and be defined as the temperature that hydrodynamic radius starts increase.Result is shown in Fig. 3.In figure 3, show the VEGFang2-0016 relative to having IHH-AAA sudden change in Fc district, without the gathering of the VEGFang2-0015 of IHH-AAA sudden change.VEGFang2-0016 shows 61 DEG C and assembles starting temperature, and shows 60 DEG C of starting temperatures without the VEGFang2-0015 of IHH-AAA sudden change.

dLS time-histories

By sample with the concentration of 1mg/mL at 20mM Histidine/L-Histidine hydrochloride, prepare in 140mMNaCl, pH6.0, to be transferred to optics 384 hole through 0.4 μm of filter plate dull and stereotyped and cover with paraffin oil by centrifugal.Sample is maintained 50 DEG C of steady temperatures until while 145 hours, by dynamic light scattering method replicate measurement hydrodynamic radius.In this experiment, natural unfolded protein gathering tendency at elevated temperature will cause median size to pass increase in time.This method based on DLS is very responsive to aggregation, because these aggregations promote scattered light intensity with super proportional manner.Even 50 DEG C (close to assembling the temperature of starting temperature, seeing above) after 145 hours, the median size increase only observing VEGFang2-0015 and VEGFang2-0016 is less than 0.5nm.

7 are stored with 100mg/mL at 40 DEG C

Sample is concentrated into final concentration 100mg/mL in 200mM argininosuccinate hydrochlorate pH5.5, sterile filtration 40 DEG C of static storages 7 days.Before and after storage, measured the content of high molecular weight species and low molecular weight species (being HMW and LMW respectively) by size exclusion chromatography method.Between the sample measured immediately after storing sample and preparation, the difference of HMW content and LMW content is reported as " HMW growth " and " LMW growth " respectively.Show result in following table and Fig. 4, these results show, and compared with VEGFang2-0016 (having IHH-AAA to suddenly change), the main peak that VEGFang2-0015 (not having IHH-AAA to suddenly change) display is larger declines and higher HMW increases.Surprisingly, compared with VEGFang2-0015 (not having IHH-AAA to suddenly change), the gathering proneness that VEGFang2-0016 (having IHH-AAA sudden change) display is lower.

Table: Δ main peak, HMW peak and LMW peak after 7 days 40 DEG C time

25 DEG C of uses or T200 instrument (GEHealthcare), the functional selection of <VEGF-ANG-2> bi-specific antibody is assessed by surperficial plasmon resonance (SPR). system is through fully setting up for studying interaction of molecules.SPR-technology is based on the specific refractory power measured near gold-plated biosensor chip surface.The change of specific refractory power represent analyte that this is injected in fixed ligand and solution on the surface interact caused by quality change.If the fixed ligand of molecule on surface is combined, then quality increases, and vice versa, when analyte dissociates (reflection complex dissociation) from fixed ligand, then and Mass lost.SPR allows continuous real-time monitoring part/analyte keying action and therefore determines association rate constant (ka), dissociation rate constant (kd) and the equilibrium constant (KD).

embodiment 3

Be combined with VEGF, ANG-2, Fc γ R and FcRn

vEGF isoform kinetics avidity, comprises evaluation cross-species reactive

By using the amine coupling reagent kit of GEHealthcare supply at pH5.0, in the upper coupling about 12 of CM5 chip (GEHealthcareBR-1005-30), capture systems (the 10 μ g/mL Goat anti human F (Ab) ' of 000 resonance units (RU) ₂; Order code: 28958325; GEHealthcareBio-SciencesAB, Sweden).Sample and system buffer liquid are that PBS-T (comprises 0.05%Tween20 ^tM10mM phosphate-buffered saline) pH7.4.Flow cell be set to 25 DEG C-and sample area group be set to 12 DEG C-and cause 2 times with running buffer.By within L/ minute, injecting 50nM solution 30 seconds with flow velocity 5 μ, catch bi-specific antibody.Continue 300 seconds by the people hVEGF121, mouse mVEGF120 or the rat rVEGF164 that within L/ minute, inject multiple concentration (start from 1:3 dilution 300nM) with flow velocity 30 μ in the solution, measure keying action.From reaching 1200 seconds mutually and starting by converting running buffer to from sample solution between monitoring.By within L/ minute, washing 60 seconds with flow velocity 30 μ with glycine pH2.1 solution, make surface regeneration.By reducing from Goat anti human F (Ab ') ₂the response value that surface obtains, revises body refractive index difference.Also reduce blank injection (=bis-reference).In order to calculate apparent K _dwith other kinetic parameters, use Lang Gemiaoer 1:1 model.Hereafter show result.

aNG-2 solution avidity, comprises evaluation cross-species reactive

By determining the concentration of free interaction mating partner in equilibrium mixture, solution avidity measures interactional avidity.Solution avidity assay method relates to and being mixed by the part (=ANG-2) of the <VEGF-ANG-2> bi-specific antibody with different concns that remain in constant density.The amine coupling reagent kit of GEHealthcare supply is used to measure the maximum possible resonance units (such as 17,000 resonance units (RU)) of the antibody be fixed on CM5 chip (GEHealthcareBR-1005-30) surface at pH5.0.Sample and system buffer liquid are HBS-PpH7.4.Flow cell be set to 25 DEG C and sample area group be set to 12 DEG C and cause 2 times with running buffer.In order to produce working curve, the ANG-2 of increasing concentration is injected the BIAcore flow cell containing immobilization <VEGF-ANG-2> bi-specific antibody.The amount of combining ANG-2 measures as resonance units (RU) and maps to concentration.It is made to reach balance in room temperature the solution (11 kinds of concentration, for <VEGF-ANG-2> bi-specific antibody, from 0 to 200nM) of often kind of part and 10nMANG-2 incubation.To dissociate ANG-2 concentration from calibrating curve determining, before and after measuring the response value with the solution of the ANG-2 of known quantity, generate described working curve.Use a model 201, use free ANG-2 concentration as y-axle and to use for the antibody concentration that suppresses as x-axle, setting up 4 parameter fittings with XLfit4 (IDBSSoftware).By determining that this point of inflexion on a curve calculates avidity.By with 0.85%H ₃pO ₄solution, with flow velocity 30 μ washing in L/ minute 1 time 30 seconds, makes surface regeneration.By reducing the response value obtained from the surface of blank coupling, revise body refractive index difference.Hereafter show result.

fcRn stable state avidity

Measure for FcRn, stable state avidity is used for mutually comparing bi-specific antibody.People FcRn is diluted in also by directional at-tachment method in coupling buffer (10 μ g/mL, sodium acetate pH5.0), use BIAcorewizard, is fixed on C1 chip (GEHealthcareBR-1005-35) to final response value 200RU.Flow cell be set to 25 DEG C and sample area group be set to 12 DEG C and cause 2 times with running buffer.Sample and system buffer liquid are that PBS-T (comprises 0.05%Tween20 ^tM10mM phosphate-buffered saline) pH6.0.In order to assess the different I gG concentration of often kind of antibody, preparation concentration 62.5nM, 125nM, 250nM and 500nM.Different sample also, in selection 180 seconds binding time situations, is expelled on chip surface by flow rate set to 30 μ L/ minute continuously.By within L/ minute, injecting PBS-TpH860 second with flow velocity 30 μ, make surface regeneration.By reducing the response value obtained from blank surface, revise body refractive index difference.Also reduce buffer injection (=bis-reference).In order to calculate stable state avidity, use the method from BIA-evaluation software.In brief, by RU value to the concentration mapping analyzed, dose-response curve is produced.Based on 2 parameter fittings, asymptotic line in calculating, thus allow determine the maximum RU value of half and therefore measure avidity.Result is shown in Fig. 5 and following table.Similarly, the avidity to cynomolgus monkey, mouse and rabbit FcRn can be determined.

fc γ RIIIa measures

Fc γ RIIIa is measured, uses direct binding assay.By using the amine coupling reagent kit of GEHealthcare supply at pH5.0, in the upper coupling about 3 of CM5 chip (GEHealthcareBR-1005-30), capture systems (the 1 μ g/mlPenta-His of 000 resonance units (RU); Qiagen).Sample and system buffer liquid are HBS-P+pH7.4.Flow cell be set to 25 DEG C-and sample area group be set to 12 DEG C-and cause 2 times with running buffer.By within L/ minute, injecting 100nM solution 60 seconds with flow velocity 5 μ, catch Fc γ RIIIa-His-acceptor.By with flow velocity 30 μ L/ minute injection 100nM bi-specific antibody or monospecific control antibodies (for IgG1 subclass and IgG4 Subclass Antibodies, anti-digoxigenin antibody (anti-Dig)) 180 seconds, measure keying action.By within L/ minute, washing 120 seconds with flow velocity 30 μ with glycine pH2.5 solution, make surface regeneration.Because Fc γ RIIIa keying action is different from Lang Gemiaoer 1:1 model, only determine combine/not combine by this assay method.In a similar manner, Fc γ RIa keying action and Fc γ RIIa keying action can be determined.Show result in Fig. 6, wherein follow-up is by introducing sudden change P329GLALA, failing the more combination to Fc γ RIII to be detected.

the independent VEGF keying action of evaluation <VEGF-ANG-2> bi-specific antibody and ANG-2 keying action

By using the amine coupling reagent kit of GEHealthcare supply at pH5.0, in the upper coupling about 3 of CM4 chip (GEHealthcareBR-1005-34), capture systems (the 10 μ g/mL Goat anti human IgG of 500 resonance units (RU); GEHealthcareBio-SciencesAB, Sweden).Sample and system buffer liquid are that PBS-T (comprises 0.05%Tween20 ^tM10mM phosphate-buffered saline) pH7.4.The temperature of flow cell is set to 25 DEG C and the temperature of sample area group is set to 12 DEG C.Before the capturing, flow cell running buffer is caused 2 times.

By within L/ minute, injecting 10nM solution 60 seconds with flow velocity 5 μ, catch bi-specific antibody.By determining successively or add the effective binding energy power of often kind of part of (flow velocity 30 μ L/ minute) simultaneously, analyze the independent keying action of often kind of part and bi-specific antibody:

1. inject people VEGF with concentration 200nM to continue 180 seconds (the single keying action of qualification antigen).

2. inject people ANG-2 with concentration 100nM to continue 180 seconds (the single keying action of qualification antigen).

3. inject people VEGF with concentration 200nM and continue 180 seconds, extra with concentration 100nM injection people lasting 180 seconds of ANG-2 (identifying the keying action of the ANG-2 under VEGF exists) subsequently.

4. inject people ANG-2 with concentration 100nM and continue 180 seconds, extra with concentration 200nM injection people VEGF (identifying the VEGF keying action under ANG-2 exists) subsequently.

5. the people VEGF of co-injection concentration 200nM and the people ANG-2 of concentration 100nM continues 180 seconds (identifying the keying action of VEGF and the keying action of ANG-2) simultaneously.

By with 3MMgCl ₂solution, with flow velocity 30 μ washing in L/ minute 60 seconds, makes surface regeneration.By reducing the response value obtained from Goat anti human IgG surface, revise body refractive index difference.

If the gained final signal of scheme 3,4 and 5 equals or is similar to the summation of independent final signal of scheme 1 and 2, then bi-specific antibody can independently of each other in conjunction with two kinds of antigens.Show result in following table, wherein two kinds of antibody VEGFang2-0016, VEGFang2-0012 all show and can be combined with VEGF and ANG-2 independently of each other.

evaluation with <VEGF-ANG-2> bi-specific antibody while VEGF keying action and aNG-2 keying action

First, by using the amine coupling reagent kit of GEHealthcare supply at pH5.0, in the upper coupling about 1 of CM4 chip (GEHealthcareBR-1005-34), the VEGF (20 μ g/mL) of 600 resonance units (RU).Sample and system buffer liquid are that PBS-T (comprises 0.05%Tween20 ^tM10mM phosphate-buffered saline) pH7.4.Flow cell be set to 25 DEG C and sample area group be set to 12 DEG C and cause 2 times with running buffer.Secondly, the bi-specific antibody solution of 50nM is injected 180 seconds with flow velocity 30 μ for L/ minute.3rd, by hANG-2 with flow velocity 30 μ injection in L/ minute 180 seconds.The combination response of hANG-2 is depended on the amount of the bi-specific antibody be combined with VEGF and shows keying action simultaneously.By with 0.85%H ₃pO ₄solution, with flow velocity 60 μ washing in L/ minute 60 seconds, makes surface regeneration.Keying action is shown the additional specificities binding signal of the <VEGF-ANG-2> bi-specific antibody that previous VEGF combines by hANG-2 simultaneously.For two kinds of bi-specific antibody VEGFang2-0015 and VEGFang2-0016, can detect and VEGF and ANG-2 of <VEGF-ANG-2> bi-specific antibody keying action (data do not show) simultaneously.

Table: result: to the kinetics avidity of the VEGF isoform from different plant species

Table: result: to the solution avidity of ANG-2

Table: result: to the avidity of the FcRn of <VEGF-ANG-2> bi-specific antibody

Table: with Fc γ RI – IIIa in conjunction with result

Table: result: the independent keying action of VEGF and ANG-2 and <VEGF-ANG-2> bi-specific antibody

embodiment 4

Mass spectroscopy

These chapters and sections describe the sign of <VEGF-ANG-2> bi-specific antibody, focus on correct assembling.By the electrospray ionization mass spectrometry (ESI-MS) of the <VEGF-ANG-2> bi-specific antibody of de-glycosylation and complete or IdeS digestion (the IgG degrading enzyme of streptococcus pyogenes (S.pyogenes)), confirm the primary structure of expection.Carry out IdeS-digestion with 100 μ g antibody purifications, described antibody and 2 μ gIdeS proteolytic enzyme (Fabricator) are at 100mmol/LNaH ₂pO ₄/ Na ₂hPO ₄, 37 DEG C of incubations 5 hours in pH7.1.Subsequently, by antibody with protein concn 1mg/mL at 100mmol/LNaH ₂pO ₄/ Na ₂hPO ₄, in pH7.1 with N-Glycosylase F, neuraminidase and O-glycosides enzyme (Roche) 37 DEG C of de-glycosylations reach 16 hours and subsequently on SephadexG25 post (GEHealthcare) by HPLC desalination.In the maXis4GUHR-QTOFMS system (BrukerDaltonik) being equipped with TriVersaNanoMate source (Advion), measure total mass by ESI-MS.

Correspond to forecast quality to the quality that the de-glycosylation (following table) of IdeS-digestion or the molecule of complete de-glycosylation (following table) obtain, described forecast quality is from by two different light chain LC _aNG-2and LC _lucentiswith two different heavy chains HC _aNG-2and HC _lucentisthe aminoacid sequence of the <VEGF-ANG-2> bi-specific antibody of composition is inferred.

Table: the quality of the dual specific <VEGF/ANG2> antibody VEGFang2-0201 (not having IHH-AAA to suddenly change) of de-glycosylation and IdeS-digestion and VEGFang2-0012 (there is IHH-AAA sudden change)

Table: the quality of deglycosylated <VEGF/ANG2> antibody VEGFang2-0016 (there is IHH-AAA sudden change) and VEGFang2-0015 (not having IHH-AAA to suddenly change)

embodiment 5

FcRn chromatogram

be coupled to Streptavidin sepharose:

By 1 gram of Streptavidin sepharose(GEHealthcare) be added into biotinylation and the acceptor of dialysis and under shake incubation two hours.By receptor-derivedization sepharosebe filled in 1mLXK post (GEHealthcare).

use the chromatogram of FcRn affinity column:

Condition:

Column dimension: 50mmx5mm

Bed height: 5cm

Carrying capacity: 50 μ g samples

Level pad: 20mMMES, containing 150mMNaCl, is adjusted to pH5.5

Elution buffer: 20mMTris/HCl, containing 150mMNaCl, is adjusted to pH8.8

Wash-out: 7.5CV level pad, to 100% elution buffer in 30CV, 10CV elution buffer

people FcRn affinity column chromatography

In the following table, the retention time of <VEGF-ANG-2> bi-specific antibody on the affinity column comprising people FcRn is provided.Use above-mentioned condition, obtain data.

Table: result: the retention time of <VEGF-ANG-2> bi-specific antibody

embodiment 6

There is pharmacokinetics (PK) characteristic of the antibody of IHH-AAA sudden change

the PK data of the genetically modified FcRn mouse of people FcRn

At life stage:

This research comprises female C57BL/6J mouse (background); FcRn defective type, but to the genetically modified mouse of people FcRn hemizygote (huFcRn, strain 276-/tg)

Part 1:

With 2 μ L Suitable solutions/animals, (namely 21 μ g compound/animals (VEGFAng2-0015 (not having IHH-AAA to suddenly change)) or 23.6 μ g compound/animals (VEGFAng2-0016 (having IHH-AAA sudden change)) inject whole mouse once in mode in vitreum in right eye.

Mouse is dispensed to 2 groups, often organizes 6 animals.Blood sample is taken from group 1 in 2,24 and 96 hours upon administration and within 7,48 and 168 hours, is taken from group 2 upon administration.

By utilizing Berlin, Germany WorldPrecisionInstruments, Inc. for receiving liter the NanoFil microsyringe system of injection, inject in the vitreum of mouse right eye.Mouse being used 2.5% isoflurane anesthesia, and in order to observe little rathole, using ratio of enlargement to be 40 times and there is the LeicaMZFL3 microscope of circular lamp of LeicaKL2500LCD flash of light.Subsequently, No. 35 pins are used to inject 2 μ L compounds.

By contraocular eyeball rear vein beard, gather blood to measure the chemical levels serum from every animal.

In room temperature after 1 hour, by within 3 minutes, obtaining at least 50 μ L serum samples from blood at 4 DEG C centrifugal (9300xg).Serum sample directly freezing after centrifugation and-80 DEG C of chilled storages until analyze.Process latter 96 hours, the treated eye of the animal of discrete group 1, and process latter 168 hours, the treated eye of the animal of discrete group 2.Sample-80 DEG C of chilled storages until analyze.

Part 2:

With 200 μ L Suitable solutions/animals (i.e. 21 μ g compound/animals (VEGFAng2-0015 (not having IHH-AAA to suddenly change)) or 23.6 μ g compound/animals (VEGFAng2-0016 (there is IHH-AAA sudden change)), with intravenously through the whole mouse of tail vein injection once.

Mouse is dispensed to 2 groups, often organizes 5 animals.Blood sample is taken from group 1 in 1,24 and 96 hour upon administration and within 7,48 and 168 hours, is taken from group 2 upon administration.By eyeball rear vein beard, gather blood to measure the chemical levels serum from every animal.

In room temperature after 1 hour, by within 3 minutes, obtaining at least 50 μ L serum samples from blood at 4 DEG C centrifugal (9300xg).Serum sample directly freezing after centrifugation and-80 DEG C of chilled storages until analyze.

the preparation of full eye lysate (mouse)

By the full eye of physical-chemical disintegration from laboratory animal, obtain eye lysate.In order to mechanical disruption, every eye is transferred to a 1.5mL conical bottom trace bottle.Freezing and after thawing, by eye with the washing of 1mL Cell Wash Buffer once (Bio-Rad, Bio-Plex cell dissolution reagent box, catalog number (Cat.No.) 171-304011).In following steps, add the Cell lysis buffer of the fresh preparation of 500 μ L and use 1.5mL to organize pestle (KimbleChase, 1.5mL pestle, production code member 749521-1500) of milling, grinding this eye.Subsequently that mixture is freezing and thaw for 5 times and again grind.In order to by lysate and residue separate tissue, by sample with 4,500g centrifugal 4 minutes.After centrifugation, supernatant collection is stored in-20 DEG C until analyze further in quantitative ELISA.

analyze

The concentration of <VEGF-ANG-2> antibody in mice serum and eye lysate is measured with enzyme-linked immunosorbent assay (ELISA).

In order to the <VEGF-ANG-2> antibody in quantitative mice serum sample and eye lysate, carry out standard solid-phase series sandwich immunoassay, use the monoclonal antibody of biotinylated and digoxigenin glycosidation as capture antibody and detect antibody.In order to the integrity of the dual specific of check analysis thing, biotinylated capture antibody identification VEGF-binding site, and the detection antibody of digoxigenin glycosidation is combined with the ANG-2 binding site of analyte.The immunocomplex of the combination of capture antibody, analyte and detection antibody in the solid phase of the microtitration flat board (SA-MTP) of Streptavidin bag quilt is detected subsequently with the horseradish peroxidase with anti-digoxigenin antibody coupling.Unconjugated material is being washed away and after adding ABTS-substrate, the signal of acquisition is directly proportional to the amount of the analyte that SA-MTP solid phase combines from SA-MTP.The measurement signal of sample is converted into concentration by the caliberator subsequently by reference to parallel analysis, carries out quantitatively.

In a first step, by SA-MTP on MTP shaking table with 500 revs/min with concentration 1 μ g/mL 100 μ L/ hole biotinylation capture antibody solution (antiidiotypic antibody mAb<Id<VEGF>Grea tT.GreaT.GTM-2.45.51-IgG-Bi (DDS)) bag by 1 hour.Meanwhile, caliberator, QC sample and sample is prepared.By caliberator and QC diluted sample to 2% serum matrix; By diluted sample until signal is in the linearity range inside of caliberator.

With capture antibody bag by SA-MTP after, flat board lavation buffer solution and 300 μ L/ holes are washed 3 times.Subsequently, 100 μ L/ hole caliberators, QC sample and sample to be pipetted on SA-MTP and with 500 revs/min of incubations 1 hour again.Analyte is combined with the solid phase of SA-MTP by capture antibody by its anti-vegf binding site now.After incubation also removes unconjugated analyte by washing flat board, antibody (antiidiotypic antibody mAb<Id-<ANG-2>Gr eatT.GreaT.GTM-2.6.81-IgG-Dig (XOSu)) is detected in the 100 μ L/ holes first of concentration 250ng/mL and is added into SA-MTP.Again, by flat board on shaking table with 500 revs/min of incubations 1 hour.After washing, the 100 μ L/ holes second of concentration 50mU/mL are detected antibody (pAb<Digoxigenin>S-Fab-POD (poly)) be added into SA-MTP hole and by flat board with 500 revs/min of incubations 1 hour again.After removing the final washing step of excess detector antibody, add 100 μ L/ holes substrate (ABTS).Antibody-enzyme conjugate catalysis the color reaction of substrate.Pass through ELISA readout instrument subsequently at 405nm wavelength (reference wavelength: 490nm ([405/490] nm)) measurement signal.

pharmacokinetic Evaluation

Use Pharmacokinetic Evaluation program WinNonlinTM (Pharsight) 5.2.1 version, calculate pharmacokinetic parameter by non-compartmental analysis.

result:

A) serum-concentration

The result of serum-concentration is shown in following table and Fig. 7 B to Fig. 7 C.

Table: VEGFAng2-0015 (not having IHH-AAA to suddenly change): in vitreum, applying and intravenously are executed addafter serum-concentrationrelatively

Table: VEGFAng2-0016 (there is IHH-AAA sudden change): in vitreum, applying and intravenously are executed addafter serum-concentrationrelatively

Table: VEGFang2-0015 (not having IHH-AAA to suddenly change) and VEGFang2-0016 (there is IHH-AAA sudden change): apply in vitreumrear each serum-concentrationcomparison)

Table: VEGFang2-0015 (not having IHH-AAA to suddenly change) and VEGFang2-0016 (there is IHH-AAA sudden change): intravenously appliesafter each serum-concentrationcomparison

result:

B) concentration in the eye lysate of left eye and right eye

The result of concentration in a lysate is shown in following table and Fig. 7 D to Fig. 7 E.

Table: VEGFang2-0015 (the not having IHH-AAA to suddenly change) concentration in eye lysate after being applied to right eye in vitreum

Table: VEGFang2-0015 (the not having IHH-AAA to suddenly change) concentration in eye lysate after intravenously applies

Table: VEGFang2-0016 (the there is IHH-AAA sudden change) concentration in eye lysate after being applied to right eye in vitreum

Table: VEGFang2-0016 (the there is IHH-AAA sudden change) concentration in eye lysate after intravenously applies

result is summed up:

After applying in vitreum, compared with the dual specific <VEGF-ANG-2> antibody VEGFang2-0015 not having IHH-AAA to suddenly change, the dual specific <VEGF-ANG-2> antibody VEGFang2-0016 (having IHH-AAA sudden change) as reported here shows similar concentration (after 96 hours and 168 hours) in eye lysate.

In addition, after applying in vitreum, compared with the dual specific <VEGF-ANG-2> antibody VEGFang2-0015 not having IHH-AAA to suddenly change, the dual specific <VEGF-ANG-2> antibody VEGFang2-0016 (having IHH-AAA sudden change) as reported here is additionally presented at scavenging(action) faster and shorter transformation period in serum.

embodiment 7

Mouse cornea micro-capsule bag vasculogenesis assay method

In order to the dual specific <VEGF-ANG-2> antibody of the ANG-2 associativity VH and VL that test the respective VEGF associativity VH and VL and SEQIDNO:28 and 29 with SEQIDNO:20 and 21 is in vivo to the blood vessel formation against function of the angiogenesis of VEGF induction, implement mouse cornea micro-capsule bag vasculogenesis assay method.In this assay method, the Nylaflo disk soaking into VEGF is implanted in avascular corneal pocket with distance fixing for limbal vascular.Blood vessel grows in cornea the VEGF gradient pointed to and formed immediately.8 to 10 week age female BAl BIc/c mouse purchased from CharlesRiver, Sulzfeld, Germany.Research approach is according to people such as Rogers, M.S., and the method that Nat.Protoc.2 (2007) 2545-2550 describes adjusts.In brief, in anesthetized mice, use knife blade and fine-pointed forceps, under the microscope according to the micro-capsule bag preparing width about 500 μm from corneal limbus to about 1mm at cornea top.The disk of implanted diameter 0.6mm ( pallCorporation, Michigan) and the surface of smooth implanted region.By disk in corresponding somatomedin or in the carrier incubation at least 30 minutes.After (or alternatively only on 3rd, 5 or 7) on the 3rd, 5 and 7, eye is taken a picture and measures vascular reaction.By calculating the percentage ratio of neovascularity area/cornea total area, by quantitative for this assay method.

Disk 300ngVEGF or PBS is loaded in contrast and implants 7.Pass in time at monitoring blood vessel on the the 3rd, 5 and/or 7 from corneal limbus to the outgrowth of disk.At the proxima luce (prox. luc) of disk implantation, by antibody using in 10mg/kg dose intravenous (apply owing to intravenously, use only because of IHH-AAA sudden change is different from VEGFang2-0016 and have mediate the identical anti-vegf of effect and the serum stable of anti-ANG-2VH and VL VEGFang2-0015 (not having IHH-AAA to suddenly change) as surrogate) to test the blood vessel formation against function to the vasculogenesis that VEGF induces in vivo.Animals received vehicle in control group.Applying volume is 10mL/kg.

embodiment 8

There is pharmacokinetics (PK) characteristic of the antibody of HHY-AAA sudden change

to the PK data of the genetically modified FcRn mouse of people FcRn

At life stage:

Part 1:

With the Suitable solutions of VEGF/ANG2-0016, VEGF/ANG2-0096, VEGF/ANG2-0098, VEGF/ANG2-0121 of 2 μ L/ animals, in right eye, inject whole mouse once in mode in vitreum.

In room temperature after 1 hour, by within 3 minutes, obtaining at least 50 μ L serum samples from blood at 4 DEG C centrifugal (9,300xg).Serum sample freezing immediately after centrifugation and-80 DEG C of chilled storages until analyze.Process latter 96 hours, the treated eye of the animal of discrete group 1, and process latter 168 hours, the treated eye of the animal of discrete group 2.Sample-80 DEG C of chilled storages until analyze.

Part 2:

With suitable VEGF/ANG2-0096, VEGF/ANG2-0098 or VEGF/ANG2-0121 of 200 μ L/ animals, with intravenous fashion through the whole mouse of tail vein injection once.

In room temperature after 1 hour, by within 3 minutes, obtaining at least 50 μ L serum samples from blood at 4 DEG C centrifugal (9,300xg).Serum sample directly freezing after centrifugation and-80 DEG C of chilled storages until analyze.

the preparation of full eye lysate (mouse)

By the full eye of physical-chemical disintegration, obtain eye lysate.In order to mechanical disruption, every eye is transferred to a 1.5mL conical bottom trace bottle.Freezing and after thawing, by eye with the washing of 1mL Cell Wash Buffer once (Bio-Rad, Bio-Plex cell dissolution reagent box, catalog number (Cat.No.) 171-304011).In following steps, add the Cell lysis buffer of the fresh preparation of 500 μ L and use 1.5mL to organize pestle (KimbleChase, 1.5mL pestle, production code member 749521-1500) of milling, grinding this eye.Subsequently that mixture is freezing and thaw for 5 times and again grind.In order to by lysate and residue separate tissue, by sample with 4,500g centrifugal 4 minutes.After centrifugation, supernatant collection is stored in-20 DEG C until analyze further in quantitative ELISA.

analyze

The concentration of antibody in mice serum and eye lysate is measured with enzyme-linked immunosorbent assay (ELISA).

In order to the antibody in quantitative mice serum sample and eye lysate, carry out standard solid-phase series sandwich immunoassay, use the monoclonal antibody of biotinylated and digoxigenin glycosidation as capture antibody and detect antibody.Specifically, in order to the integrity of the dual specific of check analysis thing, biotinylated capture antibody identification VEGF-binding site, and the detection antibody of digoxigenin glycosidation is combined with the ANG-2 binding site of analyte.The mating type immunocomplex of capture antibody, analyte and detection antibody in the solid phase of the microtitration flat board (SA-MTP) of Streptavidin bag quilt is detected subsequently with the horseradish peroxidase with anti-digoxigenin antibody coupling.Unconjugated material is being washed away and after adding ABTS-substrate, the signal of acquisition is directly proportional to the amount of the analyte that SA-MTP solid phase combines from SA-MTP.The measurement signal of sample is converted into concentration by the caliberator subsequently by reference to parallel analysis, carries out quantitatively.

In a first step, SA-MTP such as, is wrapped by 1 hour with 500 revs/min of 100 μ L/ hole biotinylation capture antibody solution (antiidiotypic antibody, mAb<Id<VEGF>Grea tT.GreaT.GTM-2.45.51-IgG-Bi (DDS)) with concentration 1 μ g/mL on MTP shaking table.Meanwhile, caliberator, QC sample and sample is prepared.By caliberator and QC diluted sample to 2% serum matrix; By diluted sample until signal is in the linearity range inside of caliberator.

Rear at SA-MTP capture antibody bag, flat board lavation buffer solution and 300 μ L/ holes are washed 3 times.Subsequently, 100 μ L/ hole caliberators, QC sample and sample to be pipetted into respectively on SA-MTP and with 500 revs/min of incubations 1 hour again.Analyte is combined with the solid phase of SA-MTP by capture antibody by one of its anti-vegf binding site now.After incubation also removes unconjugated analyte by washing flat board, the 100 μ L/ holes first of concentration 250ng/mL are detected antibody (antiidiotypic antibody, such as mAb<Id-<ANG-2>Gr eatT.GreaT.GTM-2.6.81-IgG-Dig (XOSu)) and be added into SA-MTP.Again, by flat board on shaking table with 500 revs/min of incubations 1 hour.After washing, the 100 μ L/ holes second of concentration 50mU/mL are detected antibody (such as, pAb<Digoxigenin>S-Fab-POD (poly)) be added into SA-MTP hole and by flat board with 500 revs/min of incubations 1 hour again.After removing the final washing step of excess detector antibody, add 100 μ L/ hole substrates antibody-enzyme conjugate catalysis the color reaction of substrate.By ELISA readout instrument at 405nm wavelength (reference wavelength: 490nm ([405/490] nm)) measurement signal.

pharmacokinetic Evaluation

Although for the object of clear understanding, describe aforementioned invention in detail to illustrate to a certain degree with way of example, these illustrate and example should not be construed as limiting the scope of the invention.The whole patent quoted herein and the disclosure of scientific literature is complete clearly is by reference incorporated to.

Claims

Wherein

2. IgG class Fc district according to claim 1, wherein people Fc-acceptor is people's neonatal Fc receptor (FcRn) or people Fc γ III acceptor (Fc γ RIII).

3. IgG class Fc district according to claim 2, wherein people Fc-acceptor is the newborn Fc-acceptor of people.

4. IgG class Fc district according to any one of claim 1 to 3, is wherein measured by surperficial plasmon resonance (SPR), to the avidity increase or minimizing 10% or more of people Fc-acceptor.

5. the IgG class Fc district any one of Claims 1-4, those amino-acid residues that wherein the first parent IgG class Fc district polypeptide is different from the second parent IgG class Fc district polypeptide promote the formation in different dimerization IgG class Fc district.

6. IgG class Fc district according to any one of claim 1 to 5, wherein

I) the first parent IgG class Fc district polypeptide is selected from human IgG1 Fc district polypeptide, human IgG2 Fc district polypeptide, human IgG 3Fc district polypeptide, human IgG 4Fc district polypeptide, there is sudden change L234A, the human IgG1 Fc district polypeptide of L235A, there is sudden change Y349C, T366S, L368A, the human IgG1 Fc district polypeptide of Y407V, there is sudden change L234A, L235A, Y349C, T366S, L368A, the human IgG1 Fc district polypeptide of Y407V, there is the human IgG1 Fc district polypeptide of sudden change P329G, there is sudden change L234A, L235A, the human IgG1 Fc district polypeptide of P329G, there is sudden change P329G, Y349C, T366S, L368A, the human IgG1 Fc district polypeptide of Y407V, there is sudden change L234A, L235A, P329G, Y349C, T366S, L368A, the human IgG1 Fc district polypeptide of Y407V, there is sudden change S228P, the human IgG 4Fc district polypeptide of L235E, there is sudden change S228P, L235E, the human IgG 4Fc district polypeptide of P329G, there is sudden change Y349C, T366S, L368A, the human IgG 4Fc district polypeptide of Y407V, there is sudden change S228P, L235E, Y349C, T366S, L368A, the human IgG 4Fc district polypeptide of Y407V, there is the human IgG 4Fc district polypeptide of sudden change P329G, there is sudden change P329G, Y349C, T366S, L368A, the human IgG 4Fc district polypeptide of Y407V, there is sudden change S228P, L235E, P329G, Y349C, T366S, L368A, the human IgG 4Fc district polypeptide of Y407V, there is the human IgG1 of sudden change K392D, IgG2 or IgG4 and the human IgG 3 with sudden change N392D,

And

Ii) the second parent IgG class Fc district polypeptide is selected from human IgG1 Fc district polypeptide, human IgG2 Fc district polypeptide, human IgG 3Fc district polypeptide, human IgG 4Fc district polypeptide, there is sudden change L234A, the human IgG1 Fc district polypeptide of L235A, there is sudden change S354C, the human IgG1 Fc district polypeptide of T366W, there is sudden change L234A, L235A, S354C, the human IgG1 Fc district polypeptide of T366W, there is the human IgG1 Fc district polypeptide of sudden change P329G, there is sudden change L234A, L235A, the human IgG1 Fc district polypeptide of P329G, there is sudden change P329G, S354C, the human IgG1 Fc district polypeptide of T366W, there is sudden change L234A, L235A, P329G, S354C, the human IgG1 Fc district polypeptide of T366W, there is sudden change S228P, the human IgG 4Fc district polypeptide of L235E, there is sudden change S228P, L235E, the human IgG 4Fc district polypeptide of P329G, there is sudden change S354C, the human IgG 4Fc district polypeptide of T366W, there is sudden change S228P, L235E, S354C, the human IgG 4Fc district polypeptide of T366W, there is the human IgG 4Fc district polypeptide of sudden change P329G, there is sudden change P329G, S354C, the human IgG 4Fc district polypeptide of T366W, there is sudden change S228P, L235E, P329G, S354C, the human IgG 4Fc district polypeptide of T366W, there is sudden change D399K, the human IgG1 of D356K and/or E357K and there is sudden change D399K, the human IgG2 of E356K and/or E357K, IgG3 or IgG4.

7. the IgG class Fc district any one of claim 1 to 5, wherein

And

8. the IgG class Fc district any one of claim 1 to 6, wherein

X) the first parent IgG class Fc district polypeptide has sudden change human IgG 4Fc district's polypeptide of S228P, L235E, P329G, S354C, T366W and the second parent IgG class Fc district polypeptide is the human IgG 4Fc district polypeptide with sudden change S228P, L235E, P329G, Y349C, T366S, L368A, Y407V, or

Xi) the first parent IgG class Fc district polypeptide is that to have the sudden change human IgG1 of K392D, IgG2 or IgG4Fc district polypeptide or the first parent IgG class Fc district polypeptide be have human IgG 3Fc district's polypeptide of sudden change N392D and the second parent IgG class Fc district polypeptide has human IgG1 Fc district's polypeptide of sudden change D399K, D356K and/or E357K or the second parent IgG class Fc district polypeptide is human IgG2, IgG3 or the IgG4Fc district polypeptide with sudden change D399K, E356K and/or E357K.

9. the IgG class Fc district any one of claim 1 to 5 and 7, wherein

10. the IgG class Fc district any one of claim 1 to 9, wherein different with the second variant Fc district polypeptide in 1 to 8 amino-acid residue of the first variant Fc district polypeptide except those amino-acid residues different from the second parent IgG class Fc district polypeptide except wherein the first parent IgG class Fc district polypeptide.

11. IgG class Fc districts according to claim 10, wherein different with the second variant Fc district polypeptide in 1 to 6 amino-acid residue of the first variant Fc district polypeptide except those amino-acid residues different from the second parent IgG class Fc district polypeptide except wherein the first parent IgG class Fc district polypeptide.

12. IgG class Fc districts according to claim 10, wherein different with the second variant Fc district polypeptide in 1 to 3 amino-acid residue of the first variant Fc district polypeptide except those amino-acid residues different from the second parent IgG class Fc district polypeptide except wherein the first parent IgG class Fc district polypeptide.

13. IgG class Fc districts any one of claim 1 to 12, wherein the first variant Fc district polypeptide is the 228th, 234, 235, 236, 237, 238, 239, 248, 250, 251, 252, 253, 254, 255, 256, 257, 265, 266, 267, 268, 269, 270, 272, 285, 288, 290, 291, 297, 298, 299, 307, 308, 309, 310, 311, 314, 327, 328, 329, 330, 331, 332, 385, 387, 428, 433, 434, 435 with different from the second variant Fc district polypeptide in one or more amino-acid residues at 436 (according to KabatEUindex number system numbering) places.

14. IgG class Fc districts any one of claim 1 to 2 and 4 to 13, wherein the first variant Fc district polypeptide is different from the second variant Fc district polypeptide in the 228th, 234,235,236,237,238,239,253,254,265,266,267,268,269,270,288,297,298,299,307,311,327,328,329,330,331,332,434 with one or more amino-acid residues at 435 (according to KabatEUindex number system numbering) places.

15. IgG class Fc districts any one of claim 1 to 2 and 4 to 14, wherein the first variant Fc district polypeptide is different from the second variant Fc district polypeptide in the 233rd, 236,265,297,329 with one or more amino-acid residues at 331 places.

16. IgG class Fc districts according to claim 15, wherein the first variant Fc district polypeptide because of one or more amino acid change E233P, Δ G236, D265A, N297A, N297D, P329A and P331S different from the second variant Fc district polypeptide.

17. IgG class Fc districts any one of claims 1 to 3 and 5 to 13, wherein the first variant Fc district polypeptide is different from the second variant Fc district polypeptide in the 248th, 250,251,252,253,254,255,256,257,272,285,288,290,291,308,309,310,311,314,385,386,387,428,432,433,434,435 with one or more amino-acid residues at 436 places.

18. IgG class Fc districts according to claim 17, wherein the first variant Fc district polypeptide is different with the second variant Fc district polypeptide from the one or more amino-acid residues located at the 251st, 253,310,314,432,433,435 and 436.

19. IgG class Fc districts any one of claims 1 to 3 and 5 to 18, wherein a Fc district polypeptide i) organizes I253A because being selected from, H310A and H435A, or ii) organize H310A, H433A and Y436A, or iii) organize L251D, L314D and L432D, or iv) organize L251S, one or two sudden change of L314S with L432S (according to KabatEUindex number system numbering) is different from the 2nd Fc district polypeptide, and the 2nd Fc district polypeptide is because being selected from sudden change L251D, L251S, I253A, H310A, L314D, L314S, L432D, L432S, H433A, one or two sudden change of H435A with Y436A (according to KabatEUindex number system numbering) is different from a Fc district polypeptide, thus the i) I253A that all suddenlys change, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S is contained in IgG class Fc district.

20. IgG class Fc districts any one of claims 1 to 3 and 5 to 18, wherein a Fc district polypeptide i) organizes I253A because being selected from, H310A and H435A, or ii) organize H310A, one or two sudden change of H433A with Y436A (according to KabatEUindex number system numbering) is different from the 2nd Fc district polypeptide, and the 2nd Fc district polypeptide is because being selected from sudden change I253A, H310A, H433A, one or two sudden change of H435A with Y436A (according to KabatEUindex number system numbering) is different from a Fc district polypeptide, thus the i) I253A that all suddenlys change, H310A and H435A, or ii) H310A, H433A and Y436A is contained in IgG class Fc district.

21. IgG class Fc districts any one of claims 1 to 3 and 5 to 18, sudden change I253A/H310A/H435A or H310A/H433A/Y436A or L251D/L314D/L432D or L251S/L314S/L432S or its combination (according to KabatEUindex number system numbering) is comprised in Qi Fc district, thus i) all sudden change all first or the 2nd in Fc district polypeptide, or ii) one or two sudden change in a Fc district polypeptide and one or two sudden change in the 2nd Fc district polypeptide, thus the i) I253A that all suddenlys change, H310A and H435A, or ii) H310A, H433A and Y436A, or iii) L251D, L314D and L432D, or iv) L251S, L314S and L432S is contained in IgG class Fc district.

22. IgG class Fc districts any one of claims 1 to 3 and 5 to 18, sudden change I253A/H310A/H435A or H310A/H433A/Y436A or its combination (according to KabatEUindex number system numbering) is comprised in Qi Fc district, thus i) all sudden change all first or the 2nd in Fc district polypeptide, or ii) one or two sudden change in a Fc district polypeptide and one or two sudden change in the 2nd Fc district polypeptide, thus all suddenly change i) I253A, H310A and H435A and/or ii) H310A, H433A and Y436A be contained in this IgG class Fc district.

23. IgG class Fc districts any one of claim 1 to 20, wherein compare with the IgG class Fc district of the first parent IgG class Fc district polypeptide comprised a) with the second parent IgG class Fc district polypeptide a), described IG class Fc district has the keying action to SP of reduction.

24. IgG class Fc districts any one of claim 1 to 17, it is characterized in that in Fc district, comprise sudden change M252Y/S254T/T256E (according to KabatEUindex number system numbering), thus i) all sudden change all first or the 2nd in Fc district polypeptide, or ii) one or two sudden change in a Fc district polypeptide and one or two sudden change in the 2nd Fc district polypeptide, thus all sudden change M252Y/S254T/T256E is contained in this IgG class Fc district.

25. antibody, it comprises the IgG class Fc district any one of claim 1 to 24.

26. antibody according to claim 25, wherein antibody is monoclonal antibody.

27. antibody any one of claim 25 to 26, wherein antibody is people's antibody, humanized antibody or chimeric antibody.

28. antibody any one of claim 25 to 27, wherein antibody is bi-specific antibody.

29. antibody any one of claim 25 to 28, wherein antibody is bivalent antibody.

30. antibody any one of claim 25 to 29, wherein antibody is the dual specific bivalent antibody that FcRn keying action is eliminated, and it comprises and the first antigen binding site of people VEGF specific binding and the second antigen binding site with people ANG-2 specific binding.

Wherein

γ) bi-specific antibody comprises the IgG class Fc district any one of claim 1 to 24.

32. according to the bi-specific antibody of claim 31, wherein

33.Fc district fusion polypeptide, it comprises the IgG class Fc district any one of claim 1 to 24.

34. pharmaceutical preparations, it comprises the antibody any one of claim 25 to 32 or the Fc district fusion polypeptide according to claim 33.

35. according to the pharmaceutical preparation of claim 34, and its pharmaceutical formulations is used for the treatment of Ocular Vessels disease.

36. antibody any one of claim 25 to 32 or the Fc district fusion polypeptide according to claim 33, it is used as medicine.

37. according to the purposes of claim 36, and wherein purposes is used for the treatment of Ocular Vessels disease.

38. antibody any one of claim 25 to 32 or the purposes of Fc district fusion polypeptide in manufacture medicine according to claim 33.

39. according to the purposes of claim 38, and wherein purposes is the medicine for the manufacture of being used for the treatment of Ocular Vessels disease.

40. antibody any one of claim 25 to 32 or the Fc district fusion polypeptide according to claim 33, it is used for the treatment of Ocular Vessels disease.

41. treat the method suffering from the patient of Ocular Vessels disease in the following manner: use the antibody any one of claim 25 to 32 or the Fc district fusion polypeptide according to claim 33 to needing the patient of this treatment.